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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 is a broadly and differentially expressed  SNARE protein. (A) Rabbit polyclonal (pAb) and mouse monoclonal 15G2 (mAb) antibodies recognize a major 38-kD band in  rat brain PNS. The recombinant full-length cytoplasmic domain  of syntaxin 13 (Syn13) was used as a positive control. (B) PNSs  from brain (B), heart (H), lung (Ln), kidney (K), liver (Lv),  spleen (Sp), skeletal muscle (Sk), thymus (Th), and testis (Ts)  were analyzed by Western blot using mouse monoclonal 15G2  antibody. A single major band is detected at 38 kD, with the highest expression levels in brain, spleen, lung, thymus, and testis.
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Figure 1: Syntaxin 13 is a broadly and differentially expressed SNARE protein. (A) Rabbit polyclonal (pAb) and mouse monoclonal 15G2 (mAb) antibodies recognize a major 38-kD band in rat brain PNS. The recombinant full-length cytoplasmic domain of syntaxin 13 (Syn13) was used as a positive control. (B) PNSs from brain (B), heart (H), lung (Ln), kidney (K), liver (Lv), spleen (Sp), skeletal muscle (Sk), thymus (Th), and testis (Ts) were analyzed by Western blot using mouse monoclonal 15G2 antibody. A single major band is detected at 38 kD, with the highest expression levels in brain, spleen, lung, thymus, and testis.

Mentions: Previously reported Northern blot analysis demonstrated that syntaxin 13 is expressed in all tissues with a relatively higher expression in brain, lung, spleen, and pancreas (1). To study the protein distribution we generated rabbit polyclonal and a mouse monoclonal (15G2) antibodies using the cytoplasmic portion of recombinant syntaxin 13 as antigen. Both antibodies recognized a single major 38-kD band in rat brain PNS (Fig. 1 A), which was not recognized by preimmune sera (data not shown). In some cases, a less prominent additional band of 27 kD could also be detected (Fig. 1, A and B). This band most likely represents a degradation product of full-length syntaxin 13 since it usually is not detectable in tissues that are rapidly processed for electrophoresis and becomes more prominent in samples that are slowly prepared. Both antibodies were used to analyze the distribution of syntaxin 13 protein in multiple tissues (Fig. 1 B). Syntaxin 13 was found to be expressed in all tissues, with relatively higher protein levels in brain, lung, spleen, thymus, and testes. This broad tissue distribution suggests that syntaxin 13 mediates a fundamental membrane trafficking event common to all cell types. Despite the abundant expression of syntaxin 13 in some tissues, others express relatively low levels of the protein. Perhaps functionally redundant isoforms substitute for syntaxin 13 in some cells. Alternatively, it is possible that some tissues including brain, spleen, lung, and pancreas have a prominent trafficking step mediated by syntaxin 13.


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 is a broadly and differentially expressed  SNARE protein. (A) Rabbit polyclonal (pAb) and mouse monoclonal 15G2 (mAb) antibodies recognize a major 38-kD band in  rat brain PNS. The recombinant full-length cytoplasmic domain  of syntaxin 13 (Syn13) was used as a positive control. (B) PNSs  from brain (B), heart (H), lung (Ln), kidney (K), liver (Lv),  spleen (Sp), skeletal muscle (Sk), thymus (Th), and testis (Ts)  were analyzed by Western blot using mouse monoclonal 15G2  antibody. A single major band is detected at 38 kD, with the highest expression levels in brain, spleen, lung, thymus, and testis.
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Related In: Results  -  Collection

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

Figure 1: Syntaxin 13 is a broadly and differentially expressed SNARE protein. (A) Rabbit polyclonal (pAb) and mouse monoclonal 15G2 (mAb) antibodies recognize a major 38-kD band in rat brain PNS. The recombinant full-length cytoplasmic domain of syntaxin 13 (Syn13) was used as a positive control. (B) PNSs from brain (B), heart (H), lung (Ln), kidney (K), liver (Lv), spleen (Sp), skeletal muscle (Sk), thymus (Th), and testis (Ts) were analyzed by Western blot using mouse monoclonal 15G2 antibody. A single major band is detected at 38 kD, with the highest expression levels in brain, spleen, lung, thymus, and testis.
Mentions: Previously reported Northern blot analysis demonstrated that syntaxin 13 is expressed in all tissues with a relatively higher expression in brain, lung, spleen, and pancreas (1). To study the protein distribution we generated rabbit polyclonal and a mouse monoclonal (15G2) antibodies using the cytoplasmic portion of recombinant syntaxin 13 as antigen. Both antibodies recognized a single major 38-kD band in rat brain PNS (Fig. 1 A), which was not recognized by preimmune sera (data not shown). In some cases, a less prominent additional band of 27 kD could also be detected (Fig. 1, A and B). This band most likely represents a degradation product of full-length syntaxin 13 since it usually is not detectable in tissues that are rapidly processed for electrophoresis and becomes more prominent in samples that are slowly prepared. Both antibodies were used to analyze the distribution of syntaxin 13 protein in multiple tissues (Fig. 1 B). Syntaxin 13 was found to be expressed in all tissues, with relatively higher protein levels in brain, lung, spleen, thymus, and testes. This broad tissue distribution suggests that syntaxin 13 mediates a fundamental membrane trafficking event common to all cell types. Despite the abundant expression of syntaxin 13 in some tissues, others express relatively low levels of the protein. Perhaps functionally redundant isoforms substitute for syntaxin 13 in some cells. Alternatively, it is possible that some tissues including brain, spleen, lung, and pancreas have a prominent trafficking step mediated by syntaxin 13.

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