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J Biol Chem, Vol. 273, Issue 51, 34171-34179, December 18, 1998
§,,,
From ¶ Rigel Inc., Sunnyvale, California 94086 and the
Department of Molecular and Cellular Physiology, Howard
Hughes Medical Institute, Stanford University School of Medicine,
Stanford, California 94305-5345
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Intracellular membrane traffic is thought to be
regulated in part by soluble N-ethylmaleimide-sensitive
factor-attachment protein receptors (SNAREs) through the formation of
complexes between these proteins present on vesicle and target
membranes. All known SNARE-mediated fusion events involve members of
the syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of
a large number of different syntaxin and vesicle-associated membrane
protein genes. To further investigate the spectrum of SNAREs and their
roles in membrane trafficking we characterized three novel members of
the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa)
subfamilies. The proteins are broadly expressed, suggesting a general
role in vesicle trafficking, and localize to distinct membrane
compartments. Syntaxin 8 co-localizes with markers of the endoplasmic
reticulum. Syntaxin 17, a divergent member of the syntaxin family,
partially overlaps with endoplasmic reticulum markers, and SNAP-29 is
broadly localized on multiple membranes. SNAP-29 does not contain a
predicted membrane anchor characteristic of other SNAREs. In
vitro studies established that SNAP-29 is capable of binding to a
broad range of syntaxins.
The membrane compartmentalization of cells results in the
segregation of biochemical activities through the selective transport and retention of molecules into specific organelles. As part of this
process, exo- and endocytosis regulate the availability of secreted
chemical messengers and their receptors. The organization and dynamics
of the secretory pathway are generated through a series of vesicle
budding events from donor compartments and membrane fusion events with
acceptor compartments (1). The precise molecular mechanisms responsible
for the selective capture of specific cargo into vesicles, the fidelity
of vesicle target selection, and the mechanisms of membrane fusion are
not fully understood. Contributions to understanding these processes
have come from three approaches: yeast genetics, in vitro
biochemical reconstitution of intracellular membrane trafficking, and
the study of synaptic vesicle proteins and their interactions. These
seemingly independent lines of investigation converged, leading to the
realization that a common set of molecules appear to mediate membrane
trafficking in all eucaryotic cells (2-4).
At the core of the membrane fusion process, six molecules appear to be
particularly important. In the nerve terminal, the vesicle and plasma
membrane are thought to be brought into close apposition through the
formation of a four helix bundle structure (5). A vesicle protein
VAMP,1 contributes one helix,
while the plasma membrane proteins SNAP-25 and syntaxin contribute
three helices to form a parallel bundle alternatively referred to as
the core complex, the fusion complex, or the SNARE complex (6). The
structure of this complex and the subcellular localization of the
components suggest that its formation may drive the membrane fusion
process itself, although this issue remains controversial (7). The core
complex is extremely stable and its dissociation is mediated by NSF, an
ATPase chaperone, that requires The binding of SNAREs to each other from opposite membranes may
underlie at least part of the specificity of vesicle trafficking; however, this idea has not yet been experimentally established. A
prerequisite for SNARE proteins to impart specificity to the fusion
process is that each membrane compartment should have a complement of
SNARE proteins sufficient to ensure pairing with appropriate partners.
Recently, several SNARE proteins have been characterized based either
on interactions with known SNAREs or due to sequence homology to known
SNARE proteins (12-14). Indeed, it appears that many membrane
compartments have a specific and/or unique complement of SNAREs. For
example, rsec22b, rbet1, membrin, and syntaxin 5 are localized to the
endoplasmic reticulum (ER), intermediate compartment, and the Golgi,
where they mediate trafficking at this stage of the secretory pathway
(12, 15). In contrast, syntaxin 6 is found in the
trans-Golgi network and endosomes while syntaxin 13 localizes to early and recycling endosomes (16, 17). Many other SNARE
proteins also appear to distribute uniquely within the secretory
pathway even if their precise subcellular localization is not yet known.
A complete understanding of the secretory pathway will require a full
definition of the complement of SNARE proteins in mammalian cells.
Sequence analysis of the growing family of SNAREs has already provided
important insights. In particular, while all SNARE proteins have been
proposed to be members of a large gene family, several subfamilies
exist based on higher levels of homology. While many members of the
VAMP and syntaxin families have been identified, only two closely
related members of the SNAP-25 family (SNAP-25 and SNAP-23) have been
characterized (18, 19). In this report, we present a new member of the
SNAP-25 subfamily we refer to as SNAP-29. In addition, we characterize
two new members of the syntaxin family, syntaxin 8 and syntaxin 17. These proteins are broadly expressed suggesting general roles in
membrane trafficking and, like the other SNAREs, appear to localize to
specific membrane compartments.
Isolation of SNAREs
Syntaxin 8--
In our earlier search of the
GenBankTM EST data base (20), we obtained a human
epithelial cell cDNA clone (accession number AA654455). Sequencing
of this clone and comparison to another human cDNA clone (accession
number AF062077) yielded an open reading frame (ORF) of 711 base pairs
and was designated syntaxin 8. The clone AA654455 encodes for all but
the first 8 amino acids of human syntaxin 8. AA062077 encodes for the
complete ORF of human syntaxin 8 plus 150 base pairs of 5'-untranslated region.
Syntaxin 17--
The complete coding region of human syntaxin 3 (21) was amplified in the polymerase chain reaction (PCR) and inserted
in frame with the GAL4 DNA binding domain in pAS2K, a vector derived from pAS1 (22), to create pAS/syn3. Yeast strain Y190
(CLONTECH) was sequentially transformed with first
pAS/syn3 and grown up in SD
Multiple clones contained the partial sequence of human syntaxin 17. Custom-designed oligonucleotides were used to amplify a 360-base pair
fragment using the human clone as a template for the PCR. The PCR
product was then 32P-labeled by a random hexamer priming
reaction and used to screen 6 × 105 plaques from a
rat brain 5'-Stretch PLUS cDNA library
(CLONTECH) at 61 °C. Library screening yielded
seven positive, overlapping clones. SNAP-29--
Using syntaxin 3 as a bait in the yeast-two-hybrid
system (see above), multiple clones were isolated, which contained
partial coding region of SNAP-29. Custom-designed oligonucleotides were designed and used to amplify a 390-base pair fragment taking one of the
human clones as a template for the PCR. The PCR product was used as a
probe to screen 8 × 105 plaques of a human brain
5'-Stretch PLUS cDNA library (CLONTECH) at
62 °C. The Bioinformatics
DNA and protein sequences were analyzed with the Genetics
Computer Group software package (23). The basic local alignment search
tool (BLAST) algorithm was used to search the GenBankTM
data base (24). Multiple sequence alignments were performed using the
PILEUP program using default parameters (gap creation penalty = 12 and gap extension penalty = 4), and pairwise sequence comparisons
were performed with the BESTFIT program using identical parameters.
Statistical significance of the homology of two proteins was also
determined with this program by generating z scores. z scores were calculated from the quality scores provided by
the BESTFIT program: z = (quality score of alignment in
question RNA Blot Hybridizations
Human and rat multiple tissue Northern blots were purchased from
CLONTECH. Human blots were used for syntaxin 8 and
SNAP-29, and a rat blot was used for syntaxin 17. A random-primed
32P probe was generated using full-length coding region for
each gene. Hybridization and washing were performed as described in the
manufacturer's protocol.
Expression Constructs and Transfections
The complete coding regions of syntaxin 8, syntaxin 17, and
SNAP-29 were amplified by PCR using custom-designed oligonucleotide primers with appropriate restriction sites. The PCR products were subcloned into a derivative of the pcDNA3 mammalian expression vector (Invitrogen Corp.) that contained an oligonucleotide encoding the myc epitope so that the epitope was at the
amino-terminal end of the protein.
NRK and COS cells (passage 3-20) were maintained using standard
techniques. For transfections with the expression constructs, one
barely confluent 10-cm dish of NRK cells (1 × 107
cells) was harvested with trypsin-EDTA, washed once with
phosphate-buffered saline, resuspended in 600 µl of
phosphate-buffered saline, and incubated for 10 min on ice with 10 µg
of plasmid DNA in a 0.4-cm gap electroporation cuvette (Bio-Rad). The
cells were then electroporated using 960 microfarads and 0.25 kV in a
Bio-Rad Gene Pulser with capacitance extender, seeded in appropriate
density into each well of an eight-chamber microscope slide (Nunc), and
fixed for microscopy 24 h after transfection.
COS cells were transiently transfected using the LipofectAMINE Plus
system (Life Technologies Inc.). For extraction studies, transfected
cells were harvested 48 h after transfection.
Immunofluorescence Microscopy
Indirect immunofluorescence localization was performed on NRK
and COS cells as described previously (26).
Antibodies
Monoclonal anti-myc (9E10) and rabbit
anti-myc polyclonal IgG were purchased from Santa Cruz
Biotechnology and utilized for immunofluorescence at a dilution of
1:1000 and 1:1500, respectively. For immunoblotting, the 9E10
monoclonal antibody was used at a 1:2000 dilution. For
immunofluorescence, affinity-purified anti-syntaxin 5 rabbit antibody
was used at a 1:300 dilution (26), mouse anti-transferrin receptor at a
dilution of 1:1000 (Zymed Laboratories Inc.),
polyclonal rabbit anti-BiP/GRP78 antibody at a dilution of 1:1000
(Affinity Bioreagents, Inc.), and anti-calnexin polyclonal antiserum
(27) at a dilution of 1:1500. Monoclonal antibodies against NSF were obtained from the corresponding hybridoma cell lines generated by
fusion of NS-1 mouse myeloma cells with spleen cells from BALB/c mice
immunized with His-tagged bovine NSF fusion protein (28). Ascites fluid
was produced by Josman Laboratories (Napa Valley, CA). For
immunofluorescence, ascites fluid of the anti-NSF hybridoma cell line
6A11-3 was used at a dilution of 1:1000. For Western blotting, ascites
fluid of the anti-NSF hybridoma cell line 9G7-3 was used at a dilution
of 1:2000.
In Vitro Transcription and Translation
Using the forward primers T7-hSNAP-23
(5'-TAATACGACTCACTATAGGGAGACCACATGGATGGATAATCTGTCATCAGAAG-3') and
T7-mSNAP-25
(5'-TAATACGACTCACTATAGGGAGACCACATGGATGGATGGCCGAGGACGCAGACATGCG-3') as well as the reverse primer GEX3'
(5'-GCGGGAGCTGCATGTGTCAGAGG-3'), the full-length coding region of human
SNAP-23 and mouse SNAP-25 was amplified by PCR by using the bacterial
expression constructs pGEX/hSNAP-23 and pGEX/mSNAP-25 as templates (18,
19). Full-length cDNA for SNAP-29 was linearized from the
expression construct pcDNA3 (Invitrogen). mRNA was generated by
using the T7-mMESSAGE mMACHINETM kit (Ambion Inc.), and
in vitro translations were performed in rabbit reticulocyte
lysate (Promega) supplemented with [35S]methionine (NEN
Life Science Products).
In Vitro Binding Assay
Glutathione S-transferase (GST) fusion proteins
incorporating the full cytoplasmic domains of either rat or human
syntaxins 1a, 3, 4, 7, and 13 were prepared as described (17, 29-31).
The full-length cytoplasmic domain of syntaxin 17 was prepared by PCR
using custom-designed oligonucleotide primers with appropriate restriction sites. The fragment was subcloned into pGEX-4T-1 (Qiagen), transformed into the Escherichia coli strain XL1blue-MRF,
and induced and purified as described (29). Fusion proteins were eluted
off the glutathione-agarose beads for 10 min with 25 mM reduced glutathione, 150 mM NaCl, 50 mM
Tris/HCl, pH 8.0, and excessively dialyzed against buffer A (20 mM Hepes/NaOH, pH 7.4, 150 mM NaCl, 0.05%
Tween 20).
[35S]Methionine-labeled SNAP-23/-25/-29 (14 µl of
lysate/sample) was incubated for 4 h at 4 °C with 15 µl of
solid glutathione-agarose beads and 5 µg of either syntaxin-GST
fusion protein or 25 µg of GST in 500 µl of buffer A. Samples were
washed four times in 1 ml of buffer A with brief centrifugations. The
beads were then boiled in 30 µl of SDS sample buffer containing 50 mM dithiothreitol, and eluted proteins were electrophoresed
in 12% SDS-polyacrylamide gels and processed for fluorography.
Membrane Extraction Studies
Membrane extraction studies of
myc-SNAP-29-transfected COS cells was carried out as
described previously (14, 26).
Syntaxin 8 Is a Novel Member of the Mammalian Syntaxin/SNAP-25
Family--
Searches of the data bases of expressed sequence tags
(ESTs) using the amino acid sequence of syntaxin 6 revealed the
presence of several mouse and human EST clones (AA111025, W41301, AA654455) predicted to encode a homologous protein (20). The EST clone
AA654455 was fully sequenced, and found to contain an ORF of 228 amino
acids. A BLAST search performed with this sequence identified another
human cDNA clone (AF062077), which completely overlapped with the
sequence of the clone AA654455. In addition, AF062077 provided the
nucleotide sequence of 174 nucleotides preceding the sequence of
AA654455. By assembling both sequences, a putative start Met (at
nucleotides 151-153), an open reading frame predicted to encode a
protein of 236 amino acids and a molecular mass of 26,879 Da, and a
stop codon at nucleotides 858-860 were identified (Fig.
1). When the GenBankTM
nucleotide data base was translated and searched with the predicted amino acid sequence of syntaxin 8 using the TBLASTN algorithm, significant homology to mammalian syntaxin 6 and to mammalian SNAP-25
was revealed (see Fig. 1) (13, 18). When the amino acid sequences of
these proteins were compared in a pairwise fashion using the BESTFIT
algorithm, syntaxin 8 was found to be 32% identical and 45% similar
to syntaxin 6 (z score = 23.7), when considering the
most conserved carboxyl-terminal coiled-coil domain (amino acids
138-230). By using the same algorithm, syntaxin 8 was also found to be
30% identical and 44% similar to SNAP-25 (z score = 7.8; amino acids 150-203). These results indicate an ancestral relationship between these three proteins.
Sequence analysis reveals several potential structural motifs. The
carboxyl-terminal 26 residues of syntaxin 8 probably function as a
membrane anchor because they are not charged and contain a central
stretch of hydrophobic amino acids. Syntaxin 8 is also predicted to
contain two coiled-coil domains. The first helical domain resides in
the amino-terminal part of the protein, whereas the second predicted
coiled-coil domain directly precedes the hydrophobic tail. The
amino-terminal predicted coil does not have the characteristics of a
sequence that functions in forming a core SNARE complex. For example,
the heptad repeat is not centered on a glutamine or arginine residue
that appears in all well characterized domains that associate to form
SNARE complexes. In contrast, the carboxyl-terminal repeat of syntaxin
8 aligns well with the predicted coiled-coil of syntaxin 6 and SNAP 25, and includes a conserved glutamine residue at the center of the domain.
From this analysis, we conclude that syntaxin 8 will contribute one
helical domain to the formation of a four-stranded SNARE complex.
Northern blot analysis revealed one syntaxin 8 transcript of 1.3 kb
that displayed similar expression levels across all tissues tested (see
Fig. 2A), with elevated
expression in heart. These data are consistent with a general role of
syntaxin 8 in the secretory pathway.
Intracellular targeting of a protein to a particular organelle often
provides valuable insight into its specific role in the cell. To this
end, the full-length cDNA of syntaxin 8 was tagged at its amino
terminus with the c-myc epitope and transiently expressed in
NRK cells and COS cells. myc-syntaxin 8 was detected by
indirect immunofluorescence microscopy using either the 9E10
anti-myc monoclonal antibody or affinity purified rabbit
anti-myc antibodies. As illustrated in Fig.
3 (A, C,
E, and G), myc-syntaxin 8 was
localized to intracellular membranes displaying a tubular/reticular
pattern. This staining pattern is characteristic of the ER and
overlapped significantly with that of the ER protein calnexin (Fig. 3,
E versus F and G versus H) (27). On the other hand,
myc-syntaxin 8 did not overlap with the intermediate
compartment and Golgi marker syntaxin 5 (Fig. 3, A
versus B), and the endosomal recycling pathway
marker transferrin receptor (Fig. 3, C versus
D) (21, 32). In summary, syntaxin 8 appears to be a SNARE
protein important in membrane trafficking in many different cell types,
perhaps functioning within the early secretory pathway.
Syntaxin 17 Is a Novel, Divergent Member of the Syntaxin
Family--
Using the yeast two-hybrid system (33), we isolated
multiple overlapping cDNAs, which encoded a protein that interacted with syntaxin 3. Sequencing of these cDNAs revealed a putative protein that displayed significant homology to various syntaxin family
members. A nucleic acid probe was generated from one of these clones
and used to screen a rat brain cDNA library. We obtained rat
cDNA clones containing an ORF encoding a protein of 301 amino acids
and a predicted mass of 33,182 Da (Fig.
4). Preceding the putative start Met, the
longest of the isolated cDNA clones contained 129 base pairs of
5'-untranslated region sequence. Syntaxin 7 (p = 0.0095) and syntaxin 13 (p = 0.007) are the highest
scoring mammalian proteins that are found when searching the data base with the BLAST program using the full-length amino acid sequence as a
query (14, 31). The homology between syntaxin 7, syntaxin 13, and
syntaxin 17 is illustrated in Fig. 4. The BESTFIT algorithm revealed a
35% identity and 57% similarity between syntaxin 17 and syntaxin 7 (z score = 17), which resides in the conserved membrane-proximal coiled-coil domain that defines the syntaxin family
of proteins (amino acids 174-227). Likewise, the BESTFIT algorithm
revealed a 36% identity and 55% similarity between syntaxin 17 and
syntaxin 13 (z score = 16.1; amino acids 172-227).
Syntaxin 17 also displays high homology to the yeast syntaxins Sso1
(p = 0.0041) and Sso2 (p = 0.0075)
(34), as well as to the syntaxin-related protein KNOLLE in
Arabidopsis thaliana (p = 0.0080) and a
proposed A. thaliana syntaxin T1F9.22 (AC004255)
(p = 0.0037) (35).
Unlike other members of the syntaxin family, the predicted
transmembrane domain encoded by this sequence is not at the carboxyl terminus of the ORF. Rather, it is followed by a stretch of 52 amino
acids that show no obvious homology to known proteins or motifs. A
predicted coiled-coil sequence just amino-terminal to the hydrophobic
domain fits the consensus for other syntaxins including a glutamine
residue at the center of the heptad motif.
To determine if syntaxin 17 performs a general vesicle trafficking
function in all tissues or whether it is restricted to a subset of
tissues, we determined its expression by Northern blot analysis. As
shown in Fig. 2B, syntaxin 17 is expressed in a series of
six transcripts of different sizes (2.7, 3.1, 4.0, 4.4, 6.0, and 6.4 kb). There are three major transcripts (3.1, 6.0, and 6.4 kb) in all
tissues examined; however, they are particularly abundant in liver.
Liver is well known for its prominent secretory role and is especially
enriched in the ER compared with all other cell types (36). In testis,
the 2.7- and 3.1-kb transcripts are highly expressed. It is unclear
whether the larger transcripts represent alternative splice products or
distinct, but related, sequences.
To determine the subcellular localization of syntaxin 17, we
transfected NRK cells with an epitope-tagged syntaxin 17 construct. In
immunofluorescence studies, the protein appears membrane-bound and is
localized much like syntaxin 8 to tubular/reticular structures (Fig.
5, A and C). The
enrichment of myc-syntaxin 17 in tubular/reticular structures is comparable with that of BiP (Fig. 5, A
versus B) and calnexin (Fig. 5, C
versus D) (37, 38). Whereas antibodies against
BiP preferentially stain regions of the ER surrounding the nuclear
membrane, calnexin staining overlaps with that of the BiP staining but
also extends into the more peripheral subcompartment of the ER.
Syntaxin 17 staining also extends to peripheral regions of the cells,
but its staining pattern does not precisely overlap with that of
calnexin. In comparison to calnexin, the syntaxin 17 staining appears
to localize to tubules, which are somewhat larger in size but less
dense in number than the classic reticular ER (Fig. 5, E
versus F). Thus, while the syntaxin 17 appears to be associated with a membrane compartment, perhaps a subset of the ER
such as exit or entrance sites, higher resolution studies are needed to
better understand the localization of this divergent syntaxin.
SNAP-29 Is a Novel Member of the SNAP-25 Subfamily--
In our
yeast two-hybrid system approach to identify novel proteins interacting
with syntaxin 3, we isolated the cDNA encoding a second novel
protein, which displayed homology to neuronal SNAP-25 and the
ubiquitously expressed SNAP-23 (18, 19). A complete cDNA was
isolated by screening a human brain cDNA library with the cDNA
insert of the two-hybrid clone. Sequencing revealed an ORF of 258 amino
acids with a deduced molecular mass of 28,970 Da; therefore, we termed
this clone SNAP-29. The predicted amino acid sequence of SNAP-29 as
well as an alignment of SNAP-29 with SNAP-23 and SNAP-25 protein
sequences are shown in Fig. 6. The BESTFIT algorithm revealed a 26% identity and 35% similarity between SNAP-29 and SNAP-23 (z score = 15.4; amino acids
45-242). Likewise, the BESTFIT algorithm revealed a 32% identity and
39% similarity between SNAP-29 and SNAP-25 (z score = 12.6, amino acids 53-142). The regions of highest homology of SNAP-29
to SNAP-23 and SNAP-25 reside in the amino- and carboxyl-terminal
thirds of the SNAP-29 protein, coinciding with the potential regions
that may form coiled-coil structures. Unlike the other two proteins we
characterize in this study, both of the predicted coiled-coil regions
of SNAP-29 fit the SNARE consensus suggesting that, like SNAP-23 and
25, SNAP-29 contributes two helical domains to the formation of a
four-helix bundle.
Northern blot analysis was performed to determine the tissue
distribution of SNAP-29 (Fig. 2C). Four different species of mRNA were detected in all tissues examined, with a predominant RNA
at 1.4 kb. This pattern is consistent with the notion that SNAP-29,
like SNAP-23, may participate in an event common to all cell types.
Like SNAP-23 and SNAP-25, SNAP-29 does not possess a hydrophobic
stretch of amino acids likely to form a transmembrane domain. Interestingly, unlike SNAP-23 and SNAP-25, SNAP-29 is missing the
conserved cysteine residues, found in the central part of these
proteins (see Fig. 6). In SNAP-23 and SNAP-25, one or more of these
cysteines is palmitoylated, allowing a stable membrane association of
these proteins (39). We conducted two lines of experiments to address
the question whether SNAP-29 is targeted to intracellular membrane
structures, even though it is missing a possible palmitoylation site.
First, epitope-tagged SNAP-29 was transiently expressed in NRK cells
and its subcellular localization was visualized by indirect
immunofluorescence studies. myc-SNAP-29 appears mostly
membrane-bound, but a significant portion of the protein also seems to
be of cytosolic nature (Fig. 7,
A, C, E, and G). Most of
the immunoreactivity appears to be surrounding the nuclear membrane;
however, punctate staining extending outward from juxtanuclear regions
as well as plasma membrane staining can be observed.
myc-SNAP-29 immunoreactivity partially overlaps with
syntaxin 5 (Golgi), transferrin receptor (endosomes), and lysosomal
marker lgp120 (see Fig. 7, A versus B,
C versus D, and E
versus F, respectively) (21, 32, 40).
Interestingly, the staining of SNAP-29 and the general vesicle
trafficking protein NSF coincide almost precisely (Fig. 7, G
versus H) (41). This localization indicates that
SNAP-29 is involved in multiple membrane trafficking steps (see below).
Second, we performed cell fractionation studies on COS cells
transfected with epitope-tagged SNAP-29 to investigate the nature of
the membrane association observed by indirect immunofluorescence
studies. As illustrated in Fig.
8A, a significant portion of
the protein is present in the membrane pellet of a 100,000 × g spin, which can be partially extracted by either high salt
or high pH, and even more completely by Triton X-100. Like SNAP-29, NSF
is partially extracted by high salt or high pH, but is more efficiently
extracted by detergent (Fig. 8B). Since SNAP-29 does not
contain palmitoylation sites or a stretch of hydrophobic amino acids,
the membrane association must be mediated by other membrane-bound
structures, most likely through the interaction with one or more
syntaxins (see below).
We next set out to determine if SNAP-29 recognizes syntaxin 3 exclusively or if it is capable of interacting with multiple syntaxins.
Furthermore, we asked the question if SNAP-29 displays a different
binding specificity than SNAP-23 and SNAP-25. To answer these
questions, in vitro translated SNAP-23, SNAP-25, or SNAP-29 were used to detect binding to GST-syntaxin fusion proteins. While neither protein bound to the control protein GST, both SNAP-23 and
SNAP-25 bound to GST-syntaxin 1a, GST-syntaxin 3, and GST-syntaxin 4 (Fig. 9, A-C). SNAP-29, on
the other hand, also bound with comparable affinities as SNAP-23 and
SNAP-25 to GST-syntaxin 1a, 3, and 4, confirming our two-hybrid
results, but, in addition, SNAP-29 also bound to GST-syntaxins 7, 13, and 17 (Fig. 9D). These data support the hypothesis that
SNAP-29 is bound to membrane structures via its interaction with
multiple syntaxins. In contrast to syntaxins 1a, 3, and 4, which are
localized to the plasma membrane (21), syntaxins 7, 13, and 17 are
localized to intracellular membrane structures (14, 42). The
association of epitope-tagged SNAP-29 with various intracellular
membrane structures, and the plasma membrane observed by indirect
immunofluorescence microscopy, reflects its ability to bind multiple
syntaxins as observed by our in vitro binding studies. In
summary, our data suggest that SNAP-29 is a cytoplasmic and ubiquitous
SNARE protein that is likely important in multiple membrane trafficking
steps.
The sophisticated intracellular architecture and multiple
differentiated and specialized tissues of mammals makes it likely that
a large number of mammalian v- and t-SNAREs are required for membrane
trafficking pathways. Defining the subcellular localization and tissue
distribution of novel SNAREs provides valuable insights necessary for
understanding vesicle trafficking in mammalian cells. Indeed,
definition of the number, localization, and functions of the SNAREs may
define new trafficking pathways not previously appreciated. In this
report, we characterize three novel proteins of the syntaxin/SNAP-25
gene family and study their expression patterns, subcellular
localization, and protein-protein interactions. All three proteins are
ubiquitously expressed indicating a general role in mammalian vesicle
trafficking, and each of them localizes to distinct subcellular compartments.
Syntaxin 8 is a previously uncharacterized t-SNARE, which is most
closely related to syntaxin 6, a protein implicated in
trans-Golgi network vesicle trafficking (16). Like syntaxin
6, syntaxin 8 displays significant homology to both the syntaxin and
SNAP-25 subfamilies, confirming our earlier hypothesis that these two families are themselves related (13). Fig.
10 illustrates the evolutionary
relationships among the members of this large gene family. Like a
typical syntaxin, syntaxins 6 and 8 are anchored to membranes by a
carboxyl-terminal stretch of hydrophobic amino acid residues and most
likely contribute one helical domain to the formation of a
four-stranded SNARE complex.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
SNAP to bind the core complex (8).
Dissociation of this complex allows the components to recycle for
another round of membrane fusion. Since the complex of VAMP, syntaxin,
and SNAP-25 binds to SNAP, these proteins have been referred to as
SNAP receptors or SNAREs. The sixth protein critical for membrane
fusion in the nerve terminal is nsec1 (munc-18), a member of the sec1 family (9-11). This protein binds syntaxin to form a complex distinct from the core complex, although the role of nsec1 in membrane fusion is
not clearly understood.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Trp medium (CLONTECH)
to generate Y190/syn3 strain. A combined human lymphocyte/HeLa cell
cDNA library was transformed into the Y190/syn3 strain. Eight
million transformants were placed on SDLeu/Trp/His + 40 mM 3-amino-1,2,4-triazole plates and incubated at 30 °C
for 8 days. 287 blue colonies were selected for further analysis. In
addition to SNAP, SNAP-25A, SNAP-25B, membrin, munc-18B, and Ykt6,
partial coding regions of two novel human proteins, with homology to
different syntaxins and to SNAP-25, respectively, were identified.
DNA was obtained by standard
methods, and the EcoRI fragments were subcloned into
pBluescript KS vector (Stratagene Inc.) and both strands were sequenced
using Sequenase (Amersham Pharmacia Biotech) and custom oligonucleotide
primers. The largest clone (1.7 kb) contained an 906-base pair ORF
which was designated syntaxin 17.
DNA of the four resulting positive clones was obtained by standard methods, the EcoRI fragments were
subcloned into pBluescript KS vector (Stratagene Inc.), and both
strands were sequenced using Sequenase (Amersham Pharmacia Biotech) and custom oligonucleotide primers. The largest clone (1.1 kb) contained an
777-base pair open reading frame, which was designated SNAP-29.
mean quality score of 100 alignments using randomized
sequences)/S.D. of 100 quality scores from randomized sequences. The
phylogenetic tree of the syntaxin/SNAP-25 gene family was generated by
first aligning all family members using the PILEUP program (gap
creation penalty = 1.5 and gap extension penalty = 1). A
table of the pairwise distances was then created (DISTANCES program;
Kimura method), and the GROWTREE program (UPGMA method) was used to
reconstruct the actual tree from the distance matrix. Coiled-coil
motifs were analyzed with the Coils program, version 2.1, using both
the MTIDK and the MTK matrix (25).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Syntaxin 8 is homologous to syntaxin 6 and
SNAP-25. Sequences were aligned using the Pileup program.
Identical amino acids are darkly shaded, and conserved amino
acids are lightly shaded by the Boxshade program.
Q below the sequences (residue 174 in SNAP-25) indicates the
central residue of the predicted coiled-coil. The dark
bar indicates the COOH-terminal hydrophobic region predicted
to act as a membrane anchor. Syntaxin 8 is 23% identical and 35%
similar to syntaxin 6 and 13% identical and 28% similar to SNAP-25
when compared along the whole length of the protein.
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Fig. 2.
Syntaxin 8, syntaxin 17, and SNAP-29 are
broadly and differentially expressed. Multiple-tissue Northern
blot using 32P-labeled cDNA probes. A,
syntaxin 8 transcript size is 1.3 kb. B, syntaxin 17 transcript sizes are 2.7, 3.1, 4.0, 4.4, 6.0, and 6.4 kb. C,
SNAP-29 transcript sizes are 1.4, 3.1, 3.3, and 5.2 kb. Molecular
weight markers are shown in kilobases (kb) to the left of
each panel. B, brain; H, heart; K,
kidney; Lv, liver; Ln, lung; Pn,
pancreas; Pl, placenta; Sk, skeletal muscle;
Sp, spleen; Ts, testis.
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Fig. 3.
Syntaxin 8 co-localized with ER markers.
NRK (A, B, E, and F) and
COS cells (C, D, G, and H)
were transfected with myc-tagged syntaxin 8 and then fixed,
permeabilized, and stained with the following antibodies:
anti-myc (A, C, E, and
G), anti-syntaxin 5 (Golgi) (B), anti-transferrin
receptor (endosomes) (D), and anti-calnexin (ER)
(F and H). Bar = 50 µm
(A-H).
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Fig. 4.
Syntaxin 17 is homologous to syntaxins 13 and
7. Sequences were aligned using the Pileup program. Identical
amino acids are darkly shaded, and conserved amino acids are
lightly shaded by the Boxshade program. The Q
below the sequence (residue 199 in syntaxin 7) indicates the central
residue of the predicted coiled-coil. The dark
bar indicates the COOH-terminal hydrophobic region predicted
to act as a membrane anchor. Syntaxin 17 is 16% identical and 27%
similar to syntaxin 7 and 15% identical and 30% similarity to
syntaxin 13 when compared along the whole length of the protein.
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Fig. 5.
Syntaxin 17 partially co-localizes with ER
markers. NRK cells were transfected with myc-tagged
syntaxin 17 and then fixed, permeabilized, and stained with the
following antibodies: anti-myc (A, C,
and E), anti-BiP (ER) (B), anti-calnexin (ER)
(D and F). Bar = 50 µm
(A-D) and 20 µm (E and F).
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Fig. 6.
A new SNAP SNARE: SNAP-29. Sequences
were aligned using the Pileup program. Identical amino acids are
darkly shaded, and conserved amino acids are lightly
shaded by the Boxshade program. The two Qs below the
sequences (residues 48 and 180 in SNAP23) indicate the central residues
of the two predicted coiled-coils. The asterisks (*)
indicate the cysteine residues in SNAP-23 and/or SNAP-25 that are
absent in SNAP-29. SNAP-29 is 17% identical and 31% similar to
SNAP-23 and 17% identical and 33% similar to SNAP-25 when compared
along the whole length of the protein.
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Fig. 7.
SNAP-29 is broadly localized on multiple
membranes. NRK cells were transfected with myc-tagged
SNAP-29 and then fixed, permeabilized, and stained with the following
antibodies: anti-myc (A, C,
E, and G), anti-syntaxin 5 (Golgi)
(B), anti-transferrin receptor (endosomes) (D),
anti-lgp120 (lysosomes) (F), and anti-NSF (H).
Bar = 50 µm (A-H).
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Fig. 8.
SNAP-29 is membrane-associated. COS
cells transfected with myc-SNAP-29 were homogenized and
fractioned into postnuclear (PNS), cytosolic
(Cyt) fractions, and postnuclear membrane fractions.
Membrane fractions were extracted with either control buffer
(Co), 1.5 M NaCl (NaCl), 0.2 M sodium bicarbonate at pH 11 (pH), or 2%
Triton X-100 (TX) and centrifuged at 100,000 × g. The resulting supernatant (S) and the pellet
(P) were analyzed by SDS-polyacrylamide gel electrophoresis
and immunoblotting with anti-myc (A) or anti-NSF
(B) monoclonal antibodies.
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Fig. 9.
SNAP-29 binds to multiple syntaxins in
vitro. Bacterially expressed GST or various GST-syntaxin
fusion proteins were incubated with 35S-labeled in
vitro translated SNAP-23, SNAP-25, or SNAP-29 at 4 °C, washed,
and analyzed by SDS-polyacrylamide gel electrophoresis. The gels were
stained with Coomassie Blue to confirm equal loading of various
GST-syntaxin fusion proteins (A) and were subjected to
fluorography to detect bound SNAP-23 (B), SNAP-25
(C), and SNAP-29 (D).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 10.
Evolutionary relationships of the
syntaxin/SNAP-25 gene family. A phylogenetic tree was created as
described under "Experimental Procedures." The horizontal distance
between two proteins is proportional to their evolutionary divergence.
Asterisks (*) denote proteins identified in the paper. The
proposed mode of membrane anchoring is given in the left
column: P, protein has palmitoylation sites;
TM, protein has a transmembrane anchor at its carboxyl
terminus; N, protein has neither palmitoylation sites nor a
transmembrane region. The proposed subcellular localization is given in
the right column: ER, endoplasmic
reticulum; IC, intermediate compartment; G,
Golgi; TGN, trans-Golgi network; E,
endosomal compartment; SG, secretory granules;
PM, plasma membrane. All the members of the syntaxin
subfamily have a conserved glutamine residue at the center of the
predicted carboxyl-terminal coiled-coil domain. In contrast, the
members of the SNAP-25 subfamily have two predicted coiled-coil
domains, each of them centered around a conserved glutamine residue.
We found that epitope-tagged syntaxin 8 was specifically targeted to membrane structures that also immunostained with antibodies against the ER marker calnexin, independent of cell type or expression level. At present, no mammalian syntaxin has been reported to localize specifically to the ER. Although, a longer isoform of the Golgi t-SNARE syntaxin 5 has been reported to contain an ER retrieval signal, anti-peptide antibodies recognizing specifically the NH2-terminal extension stain the Golgi apparatus and to a lesser extend the intermediate compartment (43). The simplest interpretation of our immunofluorescence studies is that syntaxin 8 functions as a vesicle trafficking protein in the early secretory pathway, possibly mediating retrograde transport from cis-Golgi membranes to the ER. Further studies are required to localize the endogenous protein at the electron microscopic level.
Although the carboxyl-terminal heptad repeat region of syntaxin 17 aligns well with the predicted carboxyl-terminal coiled-coil regions of other syntaxin subfamily members, including a conserved glutamine residue at the center of the domain, this novel syntaxin is the most distantly related member of the mammalian family (Fig. 10). Indeed, BLAST searches with the syntaxin 17 peptide sequence revealed a higher homology to the yeast syntaxins Sso1p and Sso2p than to any mammalian syntaxin (34). In addition, syntaxin 17 is related to the KNOLLE protein of Arabidopsis, a syntaxin proposed to function in cytokinesis (35, 44). Furthermore, syntaxin 17 contains a lumenal domain comprising 52 hydrophobic and hydrophilic amino acids that is absent from all other known mammalian syntaxins.
Epitope-tagged syntaxin 17 partially coincides with the ER markers BiP and calnexin, but the immunoreactivty is clearly distinct from the typical tubular-reticular pattern. Higher resolution studies will be necessary to precisely determine the compartment to which syntaxin 17 is targeted. The unusual sequence, lumenal domain, and localization pattern suggest that syntaxin 17 may function in a different type of membrane trafficking pathway from other syntaxin proteins. Perhaps like KNOLLE, syntaxin 17 is involved in processes related to cell division. For example, syntaxin 17 could be important in membrane organelle breakdown and reassembly during mitosis. Further studies are necessary to determine the role of this very unique syntaxin family member.
In contrast to the large "classical" syntaxin subfamily, the mammalian SNAP-25 subfamily has only three members identified so far, including the newly identified SNAP-29 gene. SNAP-29 is a unique SNARE with regard to its membrane interaction. Most syntaxin and VAMP proteins are anchored to a distinct membrane compartment by means of their carboxyl-terminal stretch of hydrophobic amino acid residues; for example syntaxin 8 and syntaxin 17 fall into this group. A second group of SNARE proteins have a cysteine-rich palmitoylated domain either in the central part of the protein or at the carboxyl terminus; SNAP-25, SNAP-23, and syntaxin 11 as well as a splice variant of syntaxin 2 belong to this group (14, 18, 19, 21). SNAP-29 contains neither of these membrane attachment properties. The mode of membrane anchoring of the different family members is illustrated in Fig. 10. Whereas SNAP-25 and SNAP-23 are primarily localized to the plasma membrane or upon stimulation to mast cell granules (18, 45), epitope-tagged SNAP-29 localized predominantly to intracellular membrane structures, which partially overlap with endosomal, lysosomal, and Golgi markers and significantly overlap with membrane-bound NSF. Furthermore, we found that in vitro translated SNAP-29 but not in vitro translated SNAP-23 or SNAP-25 binds to a broad range of syntaxin fusion proteins. We propose that SNAP-29, unlike SNAP-25 and SNAP-23, does not have palmitoylation sites and is therefore not anchored to a specific compartment; thus, it is capable of participating in various intracellular transport steps, interacting with different syntaxins and VAMPs, which are themselves specifically localized to a distinct membrane compartment. Considering these findings, it is likely that SNAP-29 is involved in multiple transport steps, being able to contribute two helical domains to the formation of different four-stranded SNARE complexes. Having a "general" SNAP protein participating in multiple transport steps would explain why there are only so few SNAP-25 homologs as compared with the much larger number of syntaxin and VAMP homologs.
While SNARE proteins are certainly critical mediators of membrane trafficking and fusion, their precise function is not yet defined. These proteins may mediate late steps in vesicle docking or priming, they may mediate the membrane fusion itself, or they may be involved in a combination of these processes. It is also still not clear if each vesicle trafficking step requires a unique combination of VAMP, syntaxin, and SNAP-25 proteins.
The number of coiled-coil domains that contribute to a SNARE complex
seems to be fixed. In the case of the ternary synaptic complex, the
syntaxin contributes one helical domain, which is centered on a
glutamate residue, the VAMP molecule contributes one helical domain,
which is centered on an arginine residue, and SNAP-25 contributes two
coiled-coil domains, both of them centered on a glutamate residue. In
contrast, the helical domains of four molecules (syntaxin 5, membrin,
rsec22b, and rbet1) make up the ER to Golgi SNARE complex (15). In
mammalian species, there are now approximately 10 SNAREs with a
carboxyl-terminal helical domain centered on an arginine residue, 19 SNAREs with a carboxyl-terminal helical domain centered on a glutamine
residue, and three SNAPs (SNAP-23, -25, and -29) that can each provide two helical domains with conserved glutamine residues to the formation of a SNARE complex. Thus, we expect that the approximate total number
of these proteins will be less than a factor of 2 more than those that
are now known. This number along with patterns of differential
expression in various tissues may indeed be sufficient to account for
the diversity of membrane compartments in mammalian cells. The
evolutionary relationships, the proposed mode of membrane attachment,
and the proposed subcellular localization of some of the members of the
syntaxin/SNAP-25 gene family are depicted in Fig. 10. What remains to
be determined are the binding interactions between this growing number
of SNAREs and only then will it be possible to discern if these
interaction patterns are consistent with a role in determining the
specificity of vesicle trafficking.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Jonathan Pevsner for the syntaxin 7 fusion protein construct; Carl Hurt, Rytis Prekeris, and Karen Peterson for helpful discussions; Jason Bock for help with computers; and Chris Hazuka for critical comments on the manuscript.
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FOOTNOTES |
|---|
* 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 a fellowship from the Deutsche Forschungsgemeinschaft.
To whom correspondence should be addressed. Tel.:
650-723-9075; Fax: 650-725-4436; E-mail:
scheller{at}cmgm.stanford.edu.
The abbreviations used are: VAMP, vesicle-associated membrane protein; NSF, N-ethylmaleimide-sensitive factor; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; GST, glutathione S-transferase; EST, expressed sequence tag; ORF, open reading frame; PCR, polymerase chain reaction; kb, kilobase(s); ER, endoplasmic reticulum.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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