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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 forms a complex that is regulated by NSF/ αSNAP. (A) Rat brain Triton X-100 extract was fractionated by  velocity centrifugation through 11–34% glycerol gradient. Fractions were then separated by SDS-PAGE and analyzed by immunoblotting for the presence of syntaxin 13. (B) A rat brain Triton  X-100 extract was incubated with (middle and bottom) or without  (top) recombinant NSF and αSNAP under conditions blocking  (middle) or allowing (bottom) NSF ATPase activity. Extracts  were then fractionated through 25–49% glycerol velocity gradients and sequential fractions were analyzed by immunoblotting  for the presence of syntaxin 13.
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Figure 9: Syntaxin 13 forms a complex that is regulated by NSF/ αSNAP. (A) Rat brain Triton X-100 extract was fractionated by velocity centrifugation through 11–34% glycerol gradient. Fractions were then separated by SDS-PAGE and analyzed by immunoblotting for the presence of syntaxin 13. (B) A rat brain Triton X-100 extract was incubated with (middle and bottom) or without (top) recombinant NSF and αSNAP under conditions blocking (middle) or allowing (bottom) NSF ATPase activity. Extracts were then fractionated through 25–49% glycerol velocity gradients and sequential fractions were analyzed by immunoblotting for the presence of syntaxin 13.

Mentions: The proteins shown in Fig. 9 were sequenced as described previously (9, 24). In brief, proteins immunoprecipitated from 200 ml of rat brain Triton X-100 extract were pooled, separated on SDS-PAGE, and then Coomassie stained. Protein bands were cut out, trypsin digested, and then eluted. HPLC purification of peptides and Edman microsequencing were carried out by D. Winan at the Stanford University PAN Facility.


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 forms a complex that is regulated by NSF/ αSNAP. (A) Rat brain Triton X-100 extract was fractionated by  velocity centrifugation through 11–34% glycerol gradient. Fractions were then separated by SDS-PAGE and analyzed by immunoblotting for the presence of syntaxin 13. (B) A rat brain Triton  X-100 extract was incubated with (middle and bottom) or without  (top) recombinant NSF and αSNAP under conditions blocking  (middle) or allowing (bottom) NSF ATPase activity. Extracts  were then fractionated through 25–49% glycerol velocity gradients and sequential fractions were analyzed by immunoblotting  for the presence of syntaxin 13.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132958&req=5

Figure 9: Syntaxin 13 forms a complex that is regulated by NSF/ αSNAP. (A) Rat brain Triton X-100 extract was fractionated by velocity centrifugation through 11–34% glycerol gradient. Fractions were then separated by SDS-PAGE and analyzed by immunoblotting for the presence of syntaxin 13. (B) A rat brain Triton X-100 extract was incubated with (middle and bottom) or without (top) recombinant NSF and αSNAP under conditions blocking (middle) or allowing (bottom) NSF ATPase activity. Extracts were then fractionated through 25–49% glycerol velocity gradients and sequential fractions were analyzed by immunoblotting for the presence of syntaxin 13.
Mentions: The proteins shown in Fig. 9 were sequenced as described previously (9, 24). In brief, proteins immunoprecipitated from 200 ml of rat brain Triton X-100 extract were pooled, separated on SDS-PAGE, and then Coomassie stained. Protein bands were cut out, trypsin digested, and then eluted. HPLC purification of peptides and Edman microsequencing were carried out by D. Winan at the Stanford University PAN Facility.

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