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© The Rockefeller University Press, 0021-9525/1998//957 $5.00
The Journal of Cell Biology, Volume 143, Number 4, , 1998 957-971

Regular Articles

Syntaxin 13 Mediates Cycling of Plasma Membrane Proteins via Tubulovesicular Recycling Endosomes



Rytis Prekeris*, Judith Klumperman{ddagger}, Yu A. Chen*, and Richard H. Scheller*

* Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5428; and {ddagger} Medical School, University of Utrecht, Institute for Biomembranes, 3584CX Utrecht, The Netherlands

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 βSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added {alpha}SNAP and NSF and dissociates in the presence of ATP, but not ATP{gamma}S. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins.

Key Words: vesicular transport • endosomes • protein recycling • membrane trafficking • syntaxin

Abbreviations used in this paper: BFA, brefeldin A; EE, early endosomes; LE, late endosomes; NEM, N-ethylmaleimide; NRK, normal rat kidney; NSF, N-ethylmaleimide–sensitive factor; PNS, postnuclear supernatant; RE, recycling endosomes; SLO, streptolysin-O, SNAP, soluble NSF attachment protein; SNAP-25, synaptosomal-associated protein of 25 kD; SNARE, soluble NSF attachment protein receptor; t-SNARE, target SNARE; Tf, transferrin; TfR, transferrin receptor; VAMP, vesicle-associated membrane protein.

We would like to acknowledge D. Foletti (Stanford University, Stanford, CA) for his help in culturing embryonic hippocampal neurons. We also thank M. Steegmaier (Stanford University) for assistance in hybridoma culturing and R. Winant of the Stanford PAN facility for amino acid sequencing. We thank W. Stoorvogel (University of Utrecht, Utrecht, The Netherlands) for providing useful suggestions on the recycling assay and the critical reading of the manuscript. We would also like to thank C. Hazuka for the critical reading of the manuscript. M. Niekerk, R. Scriwanek, and T. van Rijn (all from University of Utrecht) are acknowledged for the preparation of the electronmicrographs. V. Oorschot (University of Utrecht) is gratefully acknowledged for the preparation of ultrathin cryosections.


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