Localization of plasma membrane t-SNAREs syntaxin 2 and 3 in intracellular compartments
Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 2 and 3 are t-SNAREs that, according to previous over-expression studies, are predominantly localized at the plasma membrane. In the present study we investigated localization of the endogenous syntaxin 2 and 3.
Endogenous syntaxin 2 and 3 were found in NRK cells in intracellular vesicular structures in addition to regions of the plasma membrane. Treatment of these cells with N-ethylmaleimide (NEM), which is known to inactivate membrane fusion, caused syntaxin 3 to accumulate in the trans-Golgi network and syntaxin 2 in perinuclear membrane vesicles. Kinetic analysis in the presence of NEM indicated that this redistribution of syntaxin 2 and 3 takes place via actin containing structures.
Our data suggest that syntaxin 2 cycles between the plasma membrane and the perinuclear compartment whereas syntaxin 3 cycles between the plasma membrane and the trans-Golgi network. It is possible that this cycling has an important role in the regulation of t-SNARE function.
KeywordsActin Filament Actin Cytoskeleton Membrane Fusion Snare Complex National Public Health Institute
actin monomer binding protein cofilin/actin depolymerizing factor
normal rat kidney
N-ethylmaleimide-sensitive fusion factor
soluble NSF attachment proteins
vesicle-associated membrane protein
Membrane traffic is needed for the synthesis and processing of proteins and lipids as well as the maintenance of the compartmentalization of the cell. Trafficking of intracellular membranes involves the budding of vesicles from the donor membrane and the fusion of vesicles with their respective target membranes. Several proteins are involved in membrane fusion events, including the N-ethylmaleimide (NEM)-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs) and SNAP receptors (SNAREs). SNAREs are a super family of integral membrane proteins characterized by α-helical motif. The SNAREs that are functioning in neuronal exocytosis are best characterized. They include the vesicle SNARE synaptobrevin (also referred to as VAMP, vesicle-associated membrane protein) and the membrane proteins SNAP-25 and syntaxin 1 . The pairing of target SNARE (t-SNARE) with the vesicle SNARE (v-SNARE) (trans complex) pulls the membranes together and this is possibly the driving force in the mixing of the lipid bilayers. SNAREs form bundles which contain four α-helices in a parallel arrangement . In the middle of the hydrophobic bundle, there is a hydrophilic section which either contains three conserved glutamines (Q) or one conserved arginine (R). This led to the classification of SNAREs into Q-SNAREs and R-SNAREs . For instance, SNAP-25 and syntaxins are Q-SNAREs and VAMP is an R-SNARE. Three helices of the helical bundle come from Q-SNAREs and one from an R-SNARE. Syntaxins and VAMP contain one helical SNARE motif but SNAP-25 contains two motifs . The disassembly of the SNARE complexes that are formed is mediated by NSF attachment proteins, SNAPs, and the ATPase activity of NSF [1, 4].
A unique set of SNAREs is located in distinct intracellular compartments. Liposome fusion assay has demonstrated that SNAREs do show high specificity in forming complexes with each other . The formation of functional trans complexes was mostly restricted to physiologically relevant SNARE combinations. The specificity of the complex formation resides in the SNARE motifs . However, v-SNAREs are present in both anterograde and retrograde vesicles and therefore other proteins are needed to contribute to the specificity of vesicle targeting . Those proteins include inter alia small Rab GTPases, Sec1 proteins, and complexins [8, 9]. Recently it has been reported that the formation of non-cognate SNARE complexes that are non-fusogenic might have a regulatory role. These inhibitory SNAREs have been suggested to increase the specificity of membrane targeting by inhibiting membrane fusion outside their specific compartments .
Syntaxins belong to a t-SNARE family of which over a dozen have already been cloned . Over-expression studies have suggested that syntaxin 1, 2, 3, and 4 are located predominantly at the plasma membrane. Syntaxin 1 is mainly expressed in brain tissue and is thought to function specifically in neurotransmitter release, whereas syntaxin 2, 3, and 4 have a wider tissue distribution . We have previously demonstrated that syntaxin 4 is localized, in addition to the plasma membrane, in intracellular vesicular structures as well . These structures co-localized with rab11 staining. Treatment with NEM caused accumulation of syntaxin 4/rab11 positive labelling to actin filaments .
In this study, we investigated subcellular localization of endogenous syntaxin 2 and 3 in NRK cells. Similar to syntaxin 4, syntaxin 2 and 3 were found to localize in intracellular vesicular structures in addition to regions of the plasma membrane. In the case of syntaxins 2 and 3, NEM treatment resulted in the accumulation of these proteins in perinuclear membrane vesicles and the trans-Golgi network (TGN), respectively. Kinetic analysis in the presence of NEM suggested that both syntaxin 2 and 3 were redistributed to the perinuclear sites through actin containing structures.
Characterization of syntaxin 2 and 3 anti-sera
Endogenous syntaxin 2 and 3 were found to be localized in intracellular compartments
In the presence of NEM syntaxin 2 accumulates in perinuclear vesicles and syntaxin 3 in the TGN
Redistribution of syntaxin 2 and 3 takes place via actin containing structures
In the present study, we have shown that endogenous plasma membrane t-SNAREs syntaxin 2 and 3 are not exclusively localized at the plasma membrane. In addition to the plasma membrane localization, syntaxin 2 and 3 were found to localize in intracellular membrane compartments as well. Treatment with NEM caused syntaxin 2 to accumulate in perinuclear vesicular structures which partly co-localized with the transferrin receptor whereas syntaxin 3 accumulated in the TGN. It is therefore possible that syntaxin 2 might cycle between the plasma membrane and the perinuclear membrane vesicles, and syntaxin 3 between the plasma membrane and the TGN in NRK cells. Kinetic analysis suggested that actin cytoskeleton is involved in recycling of syntaxin 2 and 3 to the perinuclear sites.
Our immunofluorescence microscopy studies indicate that endogenous syntaxin 2, 3 and 4 are located only in short sections of the plasma membrane and they are not dispersed all over of the plasma membrane. Syntaxin 2, 3 and 4 co-localize with ADF, a marker for highly dynamic regions of the actin cytoskeleton. This indicates that each of these syntaxins is present in the same section of the plasma membrane and these syntaxins cycle between this active section of the plasma membrane and different intracellular sites. In previous reports it has been suggested that syntaxin 2 is present both at the apical and basolateral portion of the plasma membrane and syntaxin 3 at the apical portion of the plasma membrane in polarized cells [21, 27, 28]. Therefore it is possible that syntaxins are targeted to the active sections of the plasma membrane in non-polarized cells as well. The cycling of syntaxins would make it possible to ensure that all the components of the fusion machinery are correctly targeted to an active site in an active form.
We have previously observed that syntaxin 4 is directly associated with actin as examined by using a cosedimentation assay. Syntaxin 2 and 3, on the other hand, are not directly associated with actin . Kinetic studies indicated that syntaxin 2 and 3 transiently co-localize with actin containing filaments when transported to the perinuclear sites, whereas syntaxin 4 accumulates into actin filament like structures in the presence of NEM . These actin bundles are morphologically distinct from stress fibers and may result from altered assembly kinetics of actin filaments . Disassembly of actin fibers with cytochalasin D causes the accumulation of actin containing aggregates. Syntaxin 4 accumulates with actin into these aggregates . In contrast, syntaxin 2 and 3 stay in vesicular structures in the presence of cytochalasin D. This suggests that syntaxin 2 and 3 are not as tightly attached to actin as syntaxin 4. Interestingly, depolarization of Madin-Darby canine kidney epithelial cells (MDCK) caused relocalization of the apical and basolateral plasma membrane to functional apical and basolateral vacuoles, respectively. These vacuoles are associated with actin cytoskeleton .
In a few previous investigations endogenous intracellular syntaxin 2 or 3 labelling has also been observed. In those studies it has been suggested that syntaxin 2 and 3 have another role in intracellular membrane fusion processes besides the fusion process at the plasma membrane. It has been reported that syntaxin 2, 3 and 4 are present in phagosomal membranes and suggested that they are involved in phagosomal maturation . Similarly, syntaxin 3 was found in granular membranes of the zymogenic cells and a role of granule-granule fusion was suggested . Also in over-expression studies intracellular syntaxin labelling has been observed and it has been thought to be caused by over-expression or mislocalization . We used lysine-periodate-paraformaldehyde fixation and saponin permeabilization in our studies to preserve the intracellular vesicular structures and therefore made them more visible than if a standard paraformaldehyde fixation had been used.
The present study clearly indicates that syntaxin 2 and 3 are not solely localized at the plasma membrane but are also present in intracellular compartments and that they may cycle between these compartments and the plasma membrane through actin containing structures. This cycling of syntaxins is likely to have an important role in the regulation of t-SNARE function.
Mouse monoclonal anti-rat TGN38 antibody was a gift from Dr. G. Banting (University of Bristol, Bristol, UK.) and mouse monoclonal anti-rat transferrin receptor Ox26 hybridoma cell line was obtained from Peninsula Laboratories, Inc. (San Carlos, CA). Actin monomer binding protein cofilin/actin depolymerizing factor (ADF) guinea pig affinity purified anti-serum  was a gift from Dr. P. Lappalainen (Institute of Biothechnology, Helsinki, Finland). Plasmids encoding full length mouse syntaxin 2A and rat syntaxin 3A cDNAs in pBK-CMV vectors (Stratagen, La Jolla, CA USA) were gifts from Dr. V. Olkkonen (National Public Health Institute, Helsinki, Finland). Lissamine rhodamine (LRSC)-conjugated, Rhodamine Red™-X-conjugated and fluorescein (FITC)-conjugated secondary antibodies were purchased from Jackson Immuno Research (West Grove, PA, USA). All other reagents were of analytical grade and were obtained from commercial sources.
Production of fusion proteins and antibodies
The cytosolic domain of mouse syntaxin 2 (1–265), rat syntaxin 3 (1–263) and rat syntaxin 4 (1–272) in pGEX 2T vectors and syntaxin 2 and 3 antisera were gifts from Dr. V. Olkkonen (National Public Health Institute, Helsinki, Finland). The production and the purification of GST fusion proteins were performed according to the manufacturer's instructions (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The anti-serum for syntaxin 4-GST proteins was produced in New Zealand White rabbits.
Normal rat kidney (NRK) cells were grown at 37°C in 5% CO2 in DME supplemented with 2 mM L-glutamine, 100 U of penicillin, 10 mg/ml of streptomycin, and 10% (v/v) foetal calf serum (Biological Industries, Beit Haemek, Israel). NEM-treatment was performed by incubating cells in 1 mM NEM for 15 minutes and then further incubating in serum free DME medium.
The samples were dissolved into SDS-Laemmli buffer, separated in SDS-PAGE, and transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Nonspecific binding of antibodies was blocked with 5% fat-free milk in TBST buffer (0.15 M NaCl, 0.05% Tween 20, 10 mM Tris-HCl pH 8.0). Secondary antibodies were conjugated with either alkaline phosphatase (Sigma, St. Louis, MO, USA) or horseradish peroxidase (Bio-Rad Laboratories, Hercules, California, USA). Alkaline phosphatase and ECL reactions were performed according to the manufacturer's instructions (Promega, Madison, WI, USA and Amersham Pharmacia Biotech AB, Uppsala, Sweden, respectively).
The preparation of membrane fraction
NRK cells were grown as confluent monolayers on 10 cm dishes. The cells were washed with hypotonic swelling buffer (10 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH 7.2) and scraped in transport medium (115 mM Kacetate, 3.5 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 25 mM Hepes-KOH pH 7.4) in the presence of 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and 2 μg/ml pepstatin A. The cells were then distrupted by repeated passage through a 23-gauge needle. The homogenate was first centrifuged at 5 000 g for 20 minutes and then the supernatant was further centrifuged at 100 000 g for one hour to precipitate membranes.
NRK cells were grown as confluent monolayers on coverslips in DME. The cells were fixed with 0.08 M lysine-0.01 M periodate-2% paraformaldehyde  and permeabilized with 0.05 % saponin to maintain vesicular structures. Conventional fluorescence images were viewed using an Olympus AX70 fluorescence microscope with a SenSys CCD camera (Photometrics, Ltd., Munich, Germany). Images were converted using the Image-Pro Plus version 3.0 software (Media Cybernetics, Silver Spring, MD, USA). Confocal images were recorded using a laser scanning Leica SP1 confocal microscope. One layer was superimposed in the image.
We thank Anne Makkonen for skillful assistance. This study was supported by research grants from the Academy of Finland (E.K., A.M.B.).
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