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Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes.

Prekeris R, Klumperman J, Chen YA, Scheller RH - J. Cell Biol. (1998)

Bottom Line: Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas.This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS.These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5428, USA.

ABSTRACT
Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of betaSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

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Related in: MedlinePlus

Syntaxin 13 colocalizes with TfR. NRK cells  were fixed with 4%  paraformaldehyde, permeabilized with saponin, and  costained using affinity-purified rabbit anti-syntaxin 13  antibody (A, D, and G) and  mouse monoclonal antibodies against TfR (B), lgp120  (E), and syntaxin 6 (H). This  was followed by incubation  with Texas red-labeled anti– rabbit IgG and FITC-labeled  anti-mouse IgG antibodies  before processing for confocal microscopy. Yellow, areas  of overlap in merged images  (C, F, and I). Bars, 5 μm.
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Figure 3: Syntaxin 13 colocalizes with TfR. NRK cells were fixed with 4% paraformaldehyde, permeabilized with saponin, and costained using affinity-purified rabbit anti-syntaxin 13 antibody (A, D, and G) and mouse monoclonal antibodies against TfR (B), lgp120 (E), and syntaxin 6 (H). This was followed by incubation with Texas red-labeled anti– rabbit IgG and FITC-labeled anti-mouse IgG antibodies before processing for confocal microscopy. Yellow, areas of overlap in merged images (C, F, and I). Bars, 5 μm.

Mentions: To begin a more detailed understanding of the distribution of syntaxin 13, we compared its immunostaining with that of well-characterized markers of lysosomal, endosomal, and TGN compartments. The pattern of syntaxin 13 staining almost completely overlapped with TfR known to be enriched in RE (Fig. 3, A–C). In contrast, lgp120, a marker for late endosomes and lysosomes, showed little colocalization with syntaxin 13 (Fig. 3, D–F). In most cell types, RE are located in the peri-Golgi region, juxtaposed to the Golgi stacks. Unfortunately, light microscopy does not have the resolution necessary to differentiate Golgi, TGN, and RE compartments. Indeed, at this level of resolution, syntaxin 13 showed a significant overlap with syntaxin 6, a protein that is mainly located in the TGN (10) (Fig. 2, G–I), as well as Golgi protein mannosidase II (data not shown).


Syntaxin 13 mediates cycling of plasma membrane proteins via tubulovesicular recycling endosomes.

Prekeris R, Klumperman J, Chen YA, Scheller RH - J. Cell Biol. (1998)

Syntaxin 13 colocalizes with TfR. NRK cells  were fixed with 4%  paraformaldehyde, permeabilized with saponin, and  costained using affinity-purified rabbit anti-syntaxin 13  antibody (A, D, and G) and  mouse monoclonal antibodies against TfR (B), lgp120  (E), and syntaxin 6 (H). This  was followed by incubation  with Texas red-labeled anti– rabbit IgG and FITC-labeled  anti-mouse IgG antibodies  before processing for confocal microscopy. Yellow, areas  of overlap in merged images  (C, F, and I). Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132958&req=5

Figure 3: Syntaxin 13 colocalizes with TfR. NRK cells were fixed with 4% paraformaldehyde, permeabilized with saponin, and costained using affinity-purified rabbit anti-syntaxin 13 antibody (A, D, and G) and mouse monoclonal antibodies against TfR (B), lgp120 (E), and syntaxin 6 (H). This was followed by incubation with Texas red-labeled anti– rabbit IgG and FITC-labeled anti-mouse IgG antibodies before processing for confocal microscopy. Yellow, areas of overlap in merged images (C, F, and I). Bars, 5 μm.
Mentions: To begin a more detailed understanding of the distribution of syntaxin 13, we compared its immunostaining with that of well-characterized markers of lysosomal, endosomal, and TGN compartments. The pattern of syntaxin 13 staining almost completely overlapped with TfR known to be enriched in RE (Fig. 3, A–C). In contrast, lgp120, a marker for late endosomes and lysosomes, showed little colocalization with syntaxin 13 (Fig. 3, D–F). In most cell types, RE are located in the peri-Golgi region, juxtaposed to the Golgi stacks. Unfortunately, light microscopy does not have the resolution necessary to differentiate Golgi, TGN, and RE compartments. Indeed, at this level of resolution, syntaxin 13 showed a significant overlap with syntaxin 6, a protein that is mainly located in the TGN (10) (Fig. 2, G–I), as well as Golgi protein mannosidase II (data not shown).

Bottom Line: Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas.This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS.These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5428, USA.

ABSTRACT
Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 we examined its intracellular distribution in nonpolarized cells. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 revealed that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of betaSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

Show MeSH
Related in: MedlinePlus