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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 functions in Tf recycling in permeabilized  PC12 cells. Tf recycling was measured using SLO-permeabilized  PC12 cells as described in Materials and Methods. To label early  and recycling endosomes, PC12 cells were loaded with [125I]Tf for  30 min at 37°C. (A) Intact (open triangles) or SLO-permeabilized  PC12 cells were incubated with (closed circles) or without (open  circles) cytosol for different periods of time. Total Tf was calculated as the sum of released, glycine-extractable, and glycine-nonextractable Tf. Data shown are the means of two independent experiments. (B) SLO-permeabilized or “cracked” PC12 cells were  incubated without (C−) or with (remaining samples) cytosol for  2 h either at 4°C (4°C) or at 37°C (rest of the samples). Where indicated, cell “ghosts” were preincubated at 4°C for 1 h with either  whole molecule (anti-syn13) or Fab fragments (anti-syn13 Fab) of  anti-syntaxin 13, anti-syntaxin 5 (anti-syn5), anti-syntaxin 1A  (anti-syn1A), or rabbit IgG (IgG). In addition, where indicated,  cytosol was either incubated for 15 min on ice with 2 mM NEM  (NEM) or ATP was replaced with ATPγS (ATPγS). The data  shown are the means of three independent experiments ± SEM.  (C) SLO-permeabilized PC12 cells were preincubated with varying concentrations of anti-syntaxin 13 antibody before measuring  the release of Tf by incubation without or with cytosol for 2 h at  37°C. Data is expressed as the percentage of total cytosol dependent Tf release. The data shown are the means of three independent experiments ± SEM. Straight line, the levels of Tf release in  the absence of cytosol.
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Figure 8: Syntaxin 13 functions in Tf recycling in permeabilized PC12 cells. Tf recycling was measured using SLO-permeabilized PC12 cells as described in Materials and Methods. To label early and recycling endosomes, PC12 cells were loaded with [125I]Tf for 30 min at 37°C. (A) Intact (open triangles) or SLO-permeabilized PC12 cells were incubated with (closed circles) or without (open circles) cytosol for different periods of time. Total Tf was calculated as the sum of released, glycine-extractable, and glycine-nonextractable Tf. Data shown are the means of two independent experiments. (B) SLO-permeabilized or “cracked” PC12 cells were incubated without (C−) or with (remaining samples) cytosol for 2 h either at 4°C (4°C) or at 37°C (rest of the samples). Where indicated, cell “ghosts” were preincubated at 4°C for 1 h with either whole molecule (anti-syn13) or Fab fragments (anti-syn13 Fab) of anti-syntaxin 13, anti-syntaxin 5 (anti-syn5), anti-syntaxin 1A (anti-syn1A), or rabbit IgG (IgG). In addition, where indicated, cytosol was either incubated for 15 min on ice with 2 mM NEM (NEM) or ATP was replaced with ATPγS (ATPγS). The data shown are the means of three independent experiments ± SEM. (C) SLO-permeabilized PC12 cells were preincubated with varying concentrations of anti-syntaxin 13 antibody before measuring the release of Tf by incubation without or with cytosol for 2 h at 37°C. Data is expressed as the percentage of total cytosol dependent Tf release. The data shown are the means of three independent experiments ± SEM. Straight line, the levels of Tf release in the absence of cytosol.

Mentions: The recycling of [125I]Tf was then initiated by incubating PC12 “ghosts” at 37°C in the presence or absence of cytosol as described in Materials and Methods. Recycled Tf is expressed as a percentage of total internalized Tf. Total Tf was calculated for every sample separately, and consisted of released, glycine-extractable (plasma membrane associated), and glycine-nonextractable (internalized) Tf. Plasma membrane-associated Tf in all cases was ∼5% (data not shown). Recycling of [125I]Tf was cytosol dependent, since the absence of cytosol resulted in about twofold reduction in Tf release (Fig. 7 A). The time course of Tf recycling closely resembled the recycling in intact PC12 cells (Fig. 8 A), as well as [125I]IgG transcytosis and recycling reported in SLO-permeabilized MDCK cells expressing polymeric Ig receptor (2). The Tf recycling also required ATP and was temperature dependent (Fig. 8 B). In the samples where ATP was substituted with nonhydrolyzable ATPγS, cytosol-dependent Tf recycling was reduced to background levels. Similarly, no cytosol-dependent release of [125I]Tf was observed when samples containing cytosol and ATP were incubated at 4°C (Fig. 8 B). Moreover, NEM treatment also inhibited cytosol-dependent Tf release (Fig. 8 B). Thus, although we cannot formally exclude the possibility that NEM had its effect through proteins other then NSF, the data suggest that much of the Tf recycling as measured by this assay is mediated through SNARE-dependent fusion.


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 functions in Tf recycling in permeabilized  PC12 cells. Tf recycling was measured using SLO-permeabilized  PC12 cells as described in Materials and Methods. To label early  and recycling endosomes, PC12 cells were loaded with [125I]Tf for  30 min at 37°C. (A) Intact (open triangles) or SLO-permeabilized  PC12 cells were incubated with (closed circles) or without (open  circles) cytosol for different periods of time. Total Tf was calculated as the sum of released, glycine-extractable, and glycine-nonextractable Tf. Data shown are the means of two independent experiments. (B) SLO-permeabilized or “cracked” PC12 cells were  incubated without (C−) or with (remaining samples) cytosol for  2 h either at 4°C (4°C) or at 37°C (rest of the samples). Where indicated, cell “ghosts” were preincubated at 4°C for 1 h with either  whole molecule (anti-syn13) or Fab fragments (anti-syn13 Fab) of  anti-syntaxin 13, anti-syntaxin 5 (anti-syn5), anti-syntaxin 1A  (anti-syn1A), or rabbit IgG (IgG). In addition, where indicated,  cytosol was either incubated for 15 min on ice with 2 mM NEM  (NEM) or ATP was replaced with ATPγS (ATPγS). The data  shown are the means of three independent experiments ± SEM.  (C) SLO-permeabilized PC12 cells were preincubated with varying concentrations of anti-syntaxin 13 antibody before measuring  the release of Tf by incubation without or with cytosol for 2 h at  37°C. Data is expressed as the percentage of total cytosol dependent Tf release. The data shown are the means of three independent experiments ± SEM. Straight line, the levels of Tf release in  the absence of cytosol.
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Figure 8: Syntaxin 13 functions in Tf recycling in permeabilized PC12 cells. Tf recycling was measured using SLO-permeabilized PC12 cells as described in Materials and Methods. To label early and recycling endosomes, PC12 cells were loaded with [125I]Tf for 30 min at 37°C. (A) Intact (open triangles) or SLO-permeabilized PC12 cells were incubated with (closed circles) or without (open circles) cytosol for different periods of time. Total Tf was calculated as the sum of released, glycine-extractable, and glycine-nonextractable Tf. Data shown are the means of two independent experiments. (B) SLO-permeabilized or “cracked” PC12 cells were incubated without (C−) or with (remaining samples) cytosol for 2 h either at 4°C (4°C) or at 37°C (rest of the samples). Where indicated, cell “ghosts” were preincubated at 4°C for 1 h with either whole molecule (anti-syn13) or Fab fragments (anti-syn13 Fab) of anti-syntaxin 13, anti-syntaxin 5 (anti-syn5), anti-syntaxin 1A (anti-syn1A), or rabbit IgG (IgG). In addition, where indicated, cytosol was either incubated for 15 min on ice with 2 mM NEM (NEM) or ATP was replaced with ATPγS (ATPγS). The data shown are the means of three independent experiments ± SEM. (C) SLO-permeabilized PC12 cells were preincubated with varying concentrations of anti-syntaxin 13 antibody before measuring the release of Tf by incubation without or with cytosol for 2 h at 37°C. Data is expressed as the percentage of total cytosol dependent Tf release. The data shown are the means of three independent experiments ± SEM. Straight line, the levels of Tf release in the absence of cytosol.
Mentions: The recycling of [125I]Tf was then initiated by incubating PC12 “ghosts” at 37°C in the presence or absence of cytosol as described in Materials and Methods. Recycled Tf is expressed as a percentage of total internalized Tf. Total Tf was calculated for every sample separately, and consisted of released, glycine-extractable (plasma membrane associated), and glycine-nonextractable (internalized) Tf. Plasma membrane-associated Tf in all cases was ∼5% (data not shown). Recycling of [125I]Tf was cytosol dependent, since the absence of cytosol resulted in about twofold reduction in Tf release (Fig. 7 A). The time course of Tf recycling closely resembled the recycling in intact PC12 cells (Fig. 8 A), as well as [125I]IgG transcytosis and recycling reported in SLO-permeabilized MDCK cells expressing polymeric Ig receptor (2). The Tf recycling also required ATP and was temperature dependent (Fig. 8 B). In the samples where ATP was substituted with nonhydrolyzable ATPγS, cytosol-dependent Tf recycling was reduced to background levels. Similarly, no cytosol-dependent release of [125I]Tf was observed when samples containing cytosol and ATP were incubated at 4°C (Fig. 8 B). Moreover, NEM treatment also inhibited cytosol-dependent Tf release (Fig. 8 B). Thus, although we cannot formally exclude the possibility that NEM had its effect through proteins other then NSF, the data suggest that much of the Tf recycling as measured by this assay is mediated through SNARE-dependent fusion.

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