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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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Syntaxin 13 localizes with internalized Tf at  different temperatures. CHO  cells were incubated with  Texas Red-labeled Tf for 30  min at 37°C (A–C) or for 2.5 h  at 15°C (D–I) and then either  processed immediately (A– F) or washed and shifted to  37°C for 15 min (G–I). All  cells were then fixed with 4%  paraformaldehyde, immunostained for syntaxin 13, and  then processed for confocal  microscopy. (A, D, and G)  Syntaxin 13; (B, E, and H) Tf-Texas red; (C, F, and I)  merged images. Yellow, area  of overlap. Bars, 5 μm.
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Figure 5: Syntaxin 13 localizes with internalized Tf at different temperatures. CHO cells were incubated with Texas Red-labeled Tf for 30 min at 37°C (A–C) or for 2.5 h at 15°C (D–I) and then either processed immediately (A– F) or washed and shifted to 37°C for 15 min (G–I). All cells were then fixed with 4% paraformaldehyde, immunostained for syntaxin 13, and then processed for confocal microscopy. (A, D, and G) Syntaxin 13; (B, E, and H) Tf-Texas red; (C, F, and I) merged images. Yellow, area of overlap. Bars, 5 μm.

Mentions: In an effort to determine the functional localization of syntaxin 13, we followed the internalization and cycling of Texas red-labeled Tf (Tf-TxR) and fluorescein-labeled dextran (Dex-FITC) in CHO cells. Loading the cells with Tf-TxR at 37°C for 30 min resulted in a perinuclear staining pattern which is similar to that of Tf receptor and colocalizes extensively with syntaxin 13 (Fig. 5, A–C). When cells were incubated with Tf-TxR in the presence of 100-fold excess of unlabeled Tf, no staining was observed (data not shown) indicating the specificity of Tf-TxR uptake. Previous studies have shown that at temperatures between 15° and 22°C endocytosis does take place, but recycling is slowed, resulting in accumulation of plasma membrane proteins such as facilitative glucose transporter (GLUT4) and Tf receptor in large vesicular structures that are broadly distributed throughout the cell (50, 64). Indeed, the internalization of Tf-TxR at 15°C for 2.5 h resulted in accumulation of Tf-TxR in cytoplasmic vesicular structures (Fig. 5 E). Once again, these Tf-TxR–positive structures also immunostained with syntaxin 13 antibodies, confirming our previous results that Tf receptor and syntaxin 13 colocalizes to the same organelles (Fig. 5, D–F). Furthermore, when the 15°C block was removed by chasing the cells at 37°C for 15 min, Tf-TxR/syntaxin 13–positive structures redistributed and became indistinguishable from the previously discussed perinuclear staining pattern (Fig. 5, G–I).


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 localizes with internalized Tf at  different temperatures. CHO  cells were incubated with  Texas Red-labeled Tf for 30  min at 37°C (A–C) or for 2.5 h  at 15°C (D–I) and then either  processed immediately (A– F) or washed and shifted to  37°C for 15 min (G–I). All  cells were then fixed with 4%  paraformaldehyde, immunostained for syntaxin 13, and  then processed for confocal  microscopy. (A, D, and G)  Syntaxin 13; (B, E, and H) Tf-Texas red; (C, F, and I)  merged images. Yellow, area  of overlap. Bars, 5 μm.
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Related In: Results  -  Collection

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Figure 5: Syntaxin 13 localizes with internalized Tf at different temperatures. CHO cells were incubated with Texas Red-labeled Tf for 30 min at 37°C (A–C) or for 2.5 h at 15°C (D–I) and then either processed immediately (A– F) or washed and shifted to 37°C for 15 min (G–I). All cells were then fixed with 4% paraformaldehyde, immunostained for syntaxin 13, and then processed for confocal microscopy. (A, D, and G) Syntaxin 13; (B, E, and H) Tf-Texas red; (C, F, and I) merged images. Yellow, area of overlap. Bars, 5 μm.
Mentions: In an effort to determine the functional localization of syntaxin 13, we followed the internalization and cycling of Texas red-labeled Tf (Tf-TxR) and fluorescein-labeled dextran (Dex-FITC) in CHO cells. Loading the cells with Tf-TxR at 37°C for 30 min resulted in a perinuclear staining pattern which is similar to that of Tf receptor and colocalizes extensively with syntaxin 13 (Fig. 5, A–C). When cells were incubated with Tf-TxR in the presence of 100-fold excess of unlabeled Tf, no staining was observed (data not shown) indicating the specificity of Tf-TxR uptake. Previous studies have shown that at temperatures between 15° and 22°C endocytosis does take place, but recycling is slowed, resulting in accumulation of plasma membrane proteins such as facilitative glucose transporter (GLUT4) and Tf receptor in large vesicular structures that are broadly distributed throughout the cell (50, 64). Indeed, the internalization of Tf-TxR at 15°C for 2.5 h resulted in accumulation of Tf-TxR in cytoplasmic vesicular structures (Fig. 5 E). Once again, these Tf-TxR–positive structures also immunostained with syntaxin 13 antibodies, confirming our previous results that Tf receptor and syntaxin 13 colocalizes to the same organelles (Fig. 5, D–F). Furthermore, when the 15°C block was removed by chasing the cells at 37°C for 15 min, Tf-TxR/syntaxin 13–positive structures redistributed and became indistinguishable from the previously discussed perinuclear staining pattern (Fig. 5, G–I).

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