Syntaxin 17 is abundant in steroidogenic cells and implicated in smooth endoplasmic reticulum membrane dynamics.

The endoplasmic reticulum (ER) consists of subcompartments that have distinct protein constituents, morphological appearances, and functions. To understand the mechanisms that regulate the intricate and dynamic organization of the endoplasmic reticulum, it is important to identify and characterize the molecular machinery involved in the assembly and maintenance of the different subcompartments. Here we report that syntaxin 17 is abundantly expressed in steroidogenic cell types and specifically localizes to smooth membranes of the ER. By immunoprecipitation analyses, syntaxin 17 exists in complexes with a syntaxin regulatory protein, rsly1, and/or two intermediate compartment SNARE proteins, rsec22b and rbet1. Furthermore, we found that syntaxin 17 is anchored to the smooth endoplasmic reticulum through an unusual mechanism, requiring two adjacent hydrophobic domains near its carboxyl terminus. Converging lines of evidence indicate that syntaxin 17 functions in a vesicle-trafficking step to the smooth-surfaced tubular ER membranes that are abundant in steroidogenic cells.

nSec1 binds a closed conformation of syntaxin1A.

The Sec1 family of proteins is proposed to function in vesicle trafficking by forming complexes with target membrane SNAREs (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors) of the syntaxin family. Here, we demonstrate, by using in vitro binding assays, nondenaturing gel electrophoresis, and specific neurotoxin treatment, that the interaction of syntaxin1A with the core SNARE components, SNAP-25 (synaptosome-associated protein of 25 kD) and VAMP2 (vesicle-associated membrane protein 2), precludes the interaction with nSec1 (also called Munc18 and rbSec1). Inversely, association of nSec1 and syntaxin1A prevents assembly of the ternary SNARE complex. Furthermore, using chemical cross-linking of rat brain membranes, we identified nSec1 complexes containing syntaxin1A, but not SNAP-25 or VAMP2. These results support the hypothesis that Sec1 proteins function as syntaxin chaperons during vesicle docking, priming, and membrane fusion.

SNARE protein trafficking in polarized MDCK cells.

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains. This polarity is generated and maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways. Soluble N-ethyl maleimide-sensitive factor attachment protein receptors (SNARE) proteins of vesicle-associated membrane protein (VAMP) and syntaxin families have been suggested to play a role in the biosynthetic transport to the apical and basolateral plasma membranes of polarized cells, where they likely mediate membrane fusion. To investigate the involvement of SNARE proteins in membrane trafficking to the apical and basolateral plasma membrane in the endocytic pathway we have monitored the recycling of various VAMP and syntaxin molecules between intracellular compartments and the two plasma membrane domains in Madin-Darby canine kidney (MDCK) cells. Here we show that VAMP8/endobrevin cycles through the apical but not through the basolateral plasma membrane. Furthermore, we found that VAMP8 localizes to apical endosomal membranes in nephric tubule epithelium and in MDCK cells. This asymmetry in localization and cycling behavior suggests that VAMP8/endobrevin may play a role in apical endosomal trafficking in polarized epithelium cells.

Vesicle-associated membrane protein 4 is implicated in trans-Golgi network vesicle trafficking.

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.

SNARE interactions are not selective. Implications for membrane fusion specificity.

The SNARE hypothesis proposes that membrane trafficking specificity is mediated by preferential high affinity interactions between particular v (vesicle membrane)- and t (target membrane)-SNARE combinations. The specificity of interactions among a diverse set of SNAREs, however, is unknown. We have tested the SNARE hypothesis by analyzing potential SNARE complexes between five proteins of the vesicle-associated membrane protein (VAMP) family, three members of the synaptosome-associated protein-25 (SNAP-25) family and three members of the syntaxin family. All of the 21 combinations of SNAREs tested formed stable complexes. Sixteen were resistant to SDS denaturation, and most complexes thermally denatured between 70 and 90 degreesC. These results suggest that the specificity of membrane fusion is not encoded by the interactions between SNAREs.

Three novel proteins of the syntaxin/SNAP-25 family.

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.

Localization, dynamics, and protein interactions reveal distinct roles for ER and Golgi SNAREs.

ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.

The binding of T cell-expressed P-selectin glycoprotein ligand-1 to E- and P-selectin is differentially regulated.

The HECA452 carbohydrate epitope, also termed cutaneous lymphocyte antigen, is known to bind to E-selectin and defines a human T cell subset preferentially found in inflamed skin. Activated T cells can express a functional form of the P-selectin glycoprotein ligand-1 (PSGL-1), the major ligand known for P-selectin. Here we show that PSGL-1 can exist in two forms, of which only one carries the HECA452 epitope and binds to E-selectin, while the other only binds to P-selectin. We have analyzed the glycoprotein ligands for E- and P-selectin on the mouse CD8+ T cell clone 4G3 at 4, 8, and 12 days after antigen-specific activation. Only at day 4 did the cells bind to E-selectin, whereas cells at all three activation stages bound to P-selectin. Expression of the HECA452 epitope correlated with E-selectin binding. In affinity isolation experiments, PSGL-1 was isolated as the major ligand by E-selectin-IgG and by P-selectin-IgG; however, PSGL-1 only bound to E-selectin at day 4, whereas it bound to P-selectin at all three activation stages. Immunoprecipitated PSGL-1 from cells at day 4, but not from cells at days 8 and 12, was recognized in immunoblots by monoclonal antibody HECA452. In immunoblots of total extracts of cells at day 4, HECA452 recognized a 240/140-kDa pair of protein bands as the major antigen. These bands could be completely removed by depletion of cell extracts with anti-PSGL-1 antibodies. Our data suggest that the carbohydrate requirements for binding of PSGL-1 to P-selectin differ from those necessary for binding to E-selectin. Furthermore, we conclude that the major glycoprotein carrier for the HECA452 epitope on activated 4G3 cells is PSGL-1.

The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum.

The P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity ligand of P-selectin on myeloid cells and certain subsets of lymphoid cells. We generated the rat monoclonal antibody (MoAb) 2PH1 that recognizes an epitope within the first 19 amino acids at the N-terminus of the processed form of mouse PSGL-1. This antibody blocks attachment of mouse myeloid cells to P-selectin under both static and flow conditions. Intravenous administration of saturating amounts of 2PH1 reduced the number of rolling leukocytes in venules of the acutely exposed mouse cremaster muscle by 79% (+/-5.7%), whereas an anti-P-selectin MoAb reduced it completely. Examining the effect of the MoAb 2PH1 on the recruitment of neutrophils into chemically inflamed mouse peritoneum showed that blocking PSGL-1 inhibited neutrophil accumulation in the peritoneum by 82% (+/-7%) at 2 hours and by 59% (+/-7.9%) at 4 hours after stimulation. A similar effect was seen with the MoAb against P-selectin. Simultaneous administration of both antibodies at the 4-hour time point blocked neutrophil accumulation by 86% (+/-4.2%), arguing for an additional partner molecule for PSGL-1 besides P-selectin. This is the first demonstration of the importance of PSGL-1 in the recruitment of mouse neutrophils into inflamed tissue.

P-selectin glycoprotein ligand-1 mediates rolling of mouse bone marrow-derived mast cells on P-selectin but not efficiently on E-selectin.

It has been shown recently that mast cells play an essential role as a source of tumor necrosis factor-alpha production during neutrophil recruitment to sites of bacterial infection. Increased numbers of mast cells are indeed noted at sites of wound healing and inflammation. These cells are either recruited from the bone marrow or proliferate locally under cytokine stimulation. Little is known about how mast cell progenitors extravasate into tissue. Using antibody-like fusion proteins of mouse E-selectin and P-selectin, we have analyzed the ability of immature mouse bone marrow-derived mast cells (BMMC) to interact with the endothelial selectins. The P-selectin glycoprotein ligand-1 (PSGL-1) was affinity-isolated from detergent extracts of surface biotinylated BMMC with both selectin-IgG fusion proteins. However, only P-selectin-IgG, but not E-selectin-IgG showed significant interaction with intact BMMC as tested by flow cytometry and cell attachment assays with the immobilized fusion proteins under flow and non-flow conditions at physiological shear stress. Thus, in spite of carrying the necessary carbohydrate modifications which enable solubilized PSGL-1 to bind avidly to E-selectin, PSGL-1 on the surface of BMMC is presented in a way that prevents it from interacting efficiently with E-selectin. Affinity-purified rabbit antibodies against mouse PSGL-1 almost completely blocked the interaction of BMMC with P-selectin-IgG in flow cytometry as well as in cell adhesion assays under static and under flow conditions. Our data reveal that PSGL-1 is the major binding site for P-selectin on mouse BMMC progenitors, but does not support efficient interactions with E-selectin.

The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface.

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.

L-selectin from human, but not from mouse neutrophils binds directly to E-selectin.

L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin-Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin-Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin-Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin-Ig fusion protein was not observed. In agreement with the biochemical data, the anti-L-selectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin-Ig but not on P-selectin-Ig. No such inhibitory effect was seen with the anti-mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin-binding carbohydrate moieties are attached to different protein scaffolds in different species.

P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin.

We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin-Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin-binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.

The E-selectin-ligand ESL-1 is a variant of a receptor for fibroblast growth factor.

E-SELECTIN is an inducible cell-adhesion molecule on endothelial cells, which mediates the binding of neutrophils and functions as a Ca(2+)-dependent lectin. We have recently identified a 150K glycoprotein as the major ligand for E-selectin on myeloid cells, using a recombinant antibody-like form of mouse E-selectin as an affinity probe. Here we report the isolation of a mouse complementary DNA for this E-selectin ligand (ESL-1). The predicted amino-acid sequence of ESL-1 is 94% identical (over 1,078 amino acids) to the recently identified chicken cysteine-rich fibroblast growth-factor receptor, except for a unique 70-amino-acid aminoterminal domain of mature ESL-1. Fucosylation of ESL-1 is imperative for affinity isolation with E-selectin-IgG. A fucosylated, recombinant antibody-like form of ESL-1, but not of L-selectin, supports adhesion of E-selectin-transfected Chinese hamster ovary cells. Antibodies against ESL-1 block the binding of mouse myeloid cells to E-selectin. ESL-1, with a structure essentially identical to that of a receptor, thus functions as a cell adhesion ligand of E-selectin.