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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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Ultrathin cryosections of CHO cells showing the subcellular localization of syntaxin 13. (A and B) Syntaxin 13 (10-nm gold) is  present on extensive clusters of tubulovesicular membranes (arrowheads in A) located near Golgi (G) complex (A) or in association  with early endosomes (EE). Sometimes continuities between EE vacuoles and syntaxin 13–positive tubules are seen (arrowheads in B).  In addition, syntaxin 13 is found on EE vacuoles (B), often in parts of membrane that bear a dark cytosolic coating (arrows). (C) Double-immunogold labeling of syntaxin 13 (10-nm gold) and clathrin (15-nm gold) identifies the dark coating on the EE as clathrin. N, nucleus. Bars, 200 nm.
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Figure 6: Ultrathin cryosections of CHO cells showing the subcellular localization of syntaxin 13. (A and B) Syntaxin 13 (10-nm gold) is present on extensive clusters of tubulovesicular membranes (arrowheads in A) located near Golgi (G) complex (A) or in association with early endosomes (EE). Sometimes continuities between EE vacuoles and syntaxin 13–positive tubules are seen (arrowheads in B). In addition, syntaxin 13 is found on EE vacuoles (B), often in parts of membrane that bear a dark cytosolic coating (arrows). (C) Double-immunogold labeling of syntaxin 13 (10-nm gold) and clathrin (15-nm gold) identifies the dark coating on the EE as clathrin. N, nucleus. Bars, 200 nm.

Mentions: To assess the distribution of syntaxin 13 at the subcellular level, ultrathin cryosections were prepared from CHO cells and immunogold labeled with affinity-purified polyclonal anti-syntaxin 13 antibody. Syntaxin 13 was localized to an extensive, branched network of tubules and associated buds and vesicles through the cell (Fig. 6 A). A small portion of these membranes was surrounded by a dense cytoplasmic coating, characteristic for the presence of clathrin. Syntaxin 13–positive membranes were found near the Golgi stack (Fig. 6 A) and adjacent to vacuolar endosomes. Occasionally continuities between an endosomal vacuole and syntaxin 13–positive tubules were observed (Fig. 6 B). Quantification of the labeling pattern revealed that the vast majority (77 ± 9%) of syntaxin 13 was found on these tubules and vesicles. Additional labeling was also found at the limiting membrane of the endosomal vacuoles (13 ± 6%), of which about one-third occurred in the areas of the membrane that were coated with clathrin (Fig. 6, A and C). Label over other compartments (ER, Golgi, and plasma membrane) was around background level.


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

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

Ultrathin cryosections of CHO cells showing the subcellular localization of syntaxin 13. (A and B) Syntaxin 13 (10-nm gold) is  present on extensive clusters of tubulovesicular membranes (arrowheads in A) located near Golgi (G) complex (A) or in association  with early endosomes (EE). Sometimes continuities between EE vacuoles and syntaxin 13–positive tubules are seen (arrowheads in B).  In addition, syntaxin 13 is found on EE vacuoles (B), often in parts of membrane that bear a dark cytosolic coating (arrows). (C) Double-immunogold labeling of syntaxin 13 (10-nm gold) and clathrin (15-nm gold) identifies the dark coating on the EE as clathrin. N, nucleus. Bars, 200 nm.
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Related In: Results  -  Collection

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Figure 6: Ultrathin cryosections of CHO cells showing the subcellular localization of syntaxin 13. (A and B) Syntaxin 13 (10-nm gold) is present on extensive clusters of tubulovesicular membranes (arrowheads in A) located near Golgi (G) complex (A) or in association with early endosomes (EE). Sometimes continuities between EE vacuoles and syntaxin 13–positive tubules are seen (arrowheads in B). In addition, syntaxin 13 is found on EE vacuoles (B), often in parts of membrane that bear a dark cytosolic coating (arrows). (C) Double-immunogold labeling of syntaxin 13 (10-nm gold) and clathrin (15-nm gold) identifies the dark coating on the EE as clathrin. N, nucleus. Bars, 200 nm.
Mentions: To assess the distribution of syntaxin 13 at the subcellular level, ultrathin cryosections were prepared from CHO cells and immunogold labeled with affinity-purified polyclonal anti-syntaxin 13 antibody. Syntaxin 13 was localized to an extensive, branched network of tubules and associated buds and vesicles through the cell (Fig. 6 A). A small portion of these membranes was surrounded by a dense cytoplasmic coating, characteristic for the presence of clathrin. Syntaxin 13–positive membranes were found near the Golgi stack (Fig. 6 A) and adjacent to vacuolar endosomes. Occasionally continuities between an endosomal vacuole and syntaxin 13–positive tubules were observed (Fig. 6 B). Quantification of the labeling pattern revealed that the vast majority (77 ± 9%) of syntaxin 13 was found on these tubules and vesicles. Additional labeling was also found at the limiting membrane of the endosomal vacuoles (13 ± 6%), of which about one-third occurred in the areas of the membrane that were coated with clathrin (Fig. 6, A and C). Label over other compartments (ER, Golgi, and plasma membrane) was around background level.

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