Limits...
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.

Show MeSH

Related in: MedlinePlus

Rat brain syntaxin 13  forms a complex with βSNAP,  SNAP-25, and VAMP2/3 proteins.  Rat brain PNS was then extracted  with 1% Triton X-100 and insoluble  material was sedimented at 100,000 g  for 1 h. For immunoprecipitations  membrane extract was then preabsorbed for 2 h at 4°C with protein  A–Sepharose beads. Immunoprecipitations were carried overnight at  4°C in the presence of affinity-purified anti-syntaxin 13 rabbit polyclonal antibody coupled to protein  A–Sepharose. As a control, protein  A–Sepharose coated with IgG was  used. After the binding step the  beads were washed four times with  homogenization buffer containing  1% Triton X-100 (first three  washes) or 0.2% Triton X-100 (final wash). Washed beads were then  resuspended in SDS sample buffer  and eluted proteins separated on  SDS-PAGE. The indicated proteins  were extracted from the gel and the  amino acid sequence of peptides  determined (Table I).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132958&req=5

Figure 10: Rat brain syntaxin 13 forms a complex with βSNAP, SNAP-25, and VAMP2/3 proteins. Rat brain PNS was then extracted with 1% Triton X-100 and insoluble material was sedimented at 100,000 g for 1 h. For immunoprecipitations membrane extract was then preabsorbed for 2 h at 4°C with protein A–Sepharose beads. Immunoprecipitations were carried overnight at 4°C in the presence of affinity-purified anti-syntaxin 13 rabbit polyclonal antibody coupled to protein A–Sepharose. As a control, protein A–Sepharose coated with IgG was used. After the binding step the beads were washed four times with homogenization buffer containing 1% Triton X-100 (first three washes) or 0.2% Triton X-100 (final wash). Washed beads were then resuspended in SDS sample buffer and eluted proteins separated on SDS-PAGE. The indicated proteins were extracted from the gel and the amino acid sequence of peptides determined (Table I).

Mentions: To further investigate the mechanism of syntaxin 13 function, we attempted to purify and identify components of the 67-kD complex. Immunoprecipitations from rat brain Triton X-100 extracts using affinity-purified anti-syntaxin 13 antibodies were scaled up to obtain sufficient quantities of protein to visualize by Coomassie staining of gels (Fig. 10). Each specific protein band was digested with trypsin and subjected to Edman sequence analysis (Table I).


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

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

Rat brain syntaxin 13  forms a complex with βSNAP,  SNAP-25, and VAMP2/3 proteins.  Rat brain PNS was then extracted  with 1% Triton X-100 and insoluble  material was sedimented at 100,000 g  for 1 h. For immunoprecipitations  membrane extract was then preabsorbed for 2 h at 4°C with protein  A–Sepharose beads. Immunoprecipitations were carried overnight at  4°C in the presence of affinity-purified anti-syntaxin 13 rabbit polyclonal antibody coupled to protein  A–Sepharose. As a control, protein  A–Sepharose coated with IgG was  used. After the binding step the  beads were washed four times with  homogenization buffer containing  1% Triton X-100 (first three  washes) or 0.2% Triton X-100 (final wash). Washed beads were then  resuspended in SDS sample buffer  and eluted proteins separated on  SDS-PAGE. The indicated proteins  were extracted from the gel and the  amino acid sequence of peptides  determined (Table I).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Rat brain syntaxin 13 forms a complex with βSNAP, SNAP-25, and VAMP2/3 proteins. Rat brain PNS was then extracted with 1% Triton X-100 and insoluble material was sedimented at 100,000 g for 1 h. For immunoprecipitations membrane extract was then preabsorbed for 2 h at 4°C with protein A–Sepharose beads. Immunoprecipitations were carried overnight at 4°C in the presence of affinity-purified anti-syntaxin 13 rabbit polyclonal antibody coupled to protein A–Sepharose. As a control, protein A–Sepharose coated with IgG was used. After the binding step the beads were washed four times with homogenization buffer containing 1% Triton X-100 (first three washes) or 0.2% Triton X-100 (final wash). Washed beads were then resuspended in SDS sample buffer and eluted proteins separated on SDS-PAGE. The indicated proteins were extracted from the gel and the amino acid sequence of peptides determined (Table I).
Mentions: To further investigate the mechanism of syntaxin 13 function, we attempted to purify and identify components of the 67-kD complex. Immunoprecipitations from rat brain Triton X-100 extracts using affinity-purified anti-syntaxin 13 antibodies were scaled up to obtain sufficient quantities of protein to visualize by Coomassie staining of gels (Fig. 10). Each specific protein band was digested with trypsin and subjected to Edman sequence analysis (Table I).

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