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.
MeSH terms
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3T3 Cells
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Adenosine Triphosphate / analysis
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Animals
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Antineoplastic Agents / pharmacology
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Biological Transport / drug effects
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Biological Transport / physiology
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Brefeldin A / pharmacology
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CHO Cells
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Carrier Proteins / analysis
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Carrier Proteins / metabolism
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Cell Membrane / metabolism*
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Cell Membrane / ultrastructure
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Cricetinae
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Endosomes / metabolism*
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Endosomes / ultrastructure
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Gene Expression / physiology
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Kidney / cytology
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Ligands
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Membrane Proteins / analysis
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Mice
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Microscopy, Electron
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N-Ethylmaleimide-Sensitive Proteins
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism
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Neurons / metabolism
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Nocodazole / pharmacology
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PC12 Cells
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Protein Synthesis Inhibitors / pharmacology
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Qa-SNARE Proteins
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R-SNARE Proteins
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RNA, Messenger / analysis
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Rats
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Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
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Synaptic Vesicles / metabolism
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Synaptic Vesicles / ultrastructure
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Vesicular Transport Proteins*
Substances
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Antineoplastic Agents
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Carrier Proteins
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Ligands
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Membrane Proteins
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Nerve Tissue Proteins
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Protein Synthesis Inhibitors
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Qa-SNARE Proteins
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R-SNARE Proteins
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RNA, Messenger
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Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
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Vesicular Transport Proteins
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Brefeldin A
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Adenosine Triphosphate
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N-Ethylmaleimide-Sensitive Proteins
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Nsf protein, mouse
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Nsf protein, rat
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Nocodazole