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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 localization in different cell lines. Cells  were fixed with 4% paraformaldehyde, permeabilized with saponin, and stained using affinity-purified rabbit anti-syntaxin 13 antibody and followed by incubation with FITC-labeled anti–mouse  IgG antibodies before processing for confocal microscopy. (A)  CHO; (B) AtT-20; (C) NRK; (D) PC12; (E) NIH3T3; (F) 15 d in  vitro hippocamapal neurons. Bars: (A–E) 5 μm; (F) 10 μm.
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Figure 2: Syntaxin 13 localization in different cell lines. Cells were fixed with 4% paraformaldehyde, permeabilized with saponin, and stained using affinity-purified rabbit anti-syntaxin 13 antibody and followed by incubation with FITC-labeled anti–mouse IgG antibodies before processing for confocal microscopy. (A) CHO; (B) AtT-20; (C) NRK; (D) PC12; (E) NIH3T3; (F) 15 d in vitro hippocamapal neurons. Bars: (A–E) 5 μm; (F) 10 μm.

Mentions: Defining the localization of vesicle trafficking proteins is a critical step in understanding the specific function of the molecule. We previously reported that full-length, amino terminally c-myc epitope-tagged syntaxin 13, when transiently expressed in normal rat kidney (NRK) cells, was predominantly targeted to the post-Golgi compartment (1). To determine the localization of endogenous syntaxin 13, we stained CHO, AtT20, PC12, NRK, NIH3T3 cells, and hippocampal neurons with the affinity-purified polyclonal antibodies. Western blot analysis demonstrated that all cells used for immunofluorescence microscopy expresses syntaxin 13 (data not shown). In all nonpolarized cells syntaxin 13 was detected in small puncta throughout the cell, but primarily located in juctanuclear area (Fig. 2, A–E). Interestingly, in hippocampal neurons syntaxin 13 immunoreactive organelles were scattered throughout dendritic and axonal processes (Fig. 2 F), as identified by costaining with anti-MAP2 (dendritic marker) and anti-SV2a (axonal marker) antibodies (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 localization in different cell lines. Cells  were fixed with 4% paraformaldehyde, permeabilized with saponin, and stained using affinity-purified rabbit anti-syntaxin 13 antibody and followed by incubation with FITC-labeled anti–mouse  IgG antibodies before processing for confocal microscopy. (A)  CHO; (B) AtT-20; (C) NRK; (D) PC12; (E) NIH3T3; (F) 15 d in  vitro hippocamapal neurons. Bars: (A–E) 5 μm; (F) 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Syntaxin 13 localization in different cell lines. Cells were fixed with 4% paraformaldehyde, permeabilized with saponin, and stained using affinity-purified rabbit anti-syntaxin 13 antibody and followed by incubation with FITC-labeled anti–mouse IgG antibodies before processing for confocal microscopy. (A) CHO; (B) AtT-20; (C) NRK; (D) PC12; (E) NIH3T3; (F) 15 d in vitro hippocamapal neurons. Bars: (A–E) 5 μm; (F) 10 μm.
Mentions: Defining the localization of vesicle trafficking proteins is a critical step in understanding the specific function of the molecule. We previously reported that full-length, amino terminally c-myc epitope-tagged syntaxin 13, when transiently expressed in normal rat kidney (NRK) cells, was predominantly targeted to the post-Golgi compartment (1). To determine the localization of endogenous syntaxin 13, we stained CHO, AtT20, PC12, NRK, NIH3T3 cells, and hippocampal neurons with the affinity-purified polyclonal antibodies. Western blot analysis demonstrated that all cells used for immunofluorescence microscopy expresses syntaxin 13 (data not shown). In all nonpolarized cells syntaxin 13 was detected in small puncta throughout the cell, but primarily located in juctanuclear area (Fig. 2, A–E). Interestingly, in hippocampal neurons syntaxin 13 immunoreactive organelles were scattered throughout dendritic and axonal processes (Fig. 2 F), as identified by costaining with anti-MAP2 (dendritic marker) and anti-SV2a (axonal marker) antibodies (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