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© The Rockefeller University Press, 0021-9525/1999//869 $5.00
The Journal of Cell Biology, Volume 144, Number 5, , 1999 869-881

Regular Articles

SNARE Membrane Trafficking Dynamics In Vivo



Daniel S. Chao*, Jesse C. Hay*, Shawn Winnick*, Rytis Prekeris*, Judith Klumperman{ddagger}, 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, Institute for Biomembranes, University of Utrecht, 3584CX Utrecht, The Netherlands

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in ~1-µm cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at ~1-µm ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched ~1-µm peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.

Key Words: vesicle trafficking • fluorescent proteins • SNAREs • membrane proteins • endoplasmic reticulum

Abbreviations used in this paper: B,C,G,Y FP, blue, cyan, green, yellow fluorescent protein; IC, intermediate compartment; NRK, normal rat kidney; NSF, N-ethylmaleimide-sensitive factor; SNAP-25, synaptosome-associated protein of 25 kD; SNAP, soluble NSF attachment protein; SNARE, soluble NSF attachment protein receptor; VAMP, vesicle-associated membrane protein; ts-G-GFP, temperature sensitive vesicular stomatitis virus-associated membrane protein; VTC, vesicular tubular cluster.

Address correspondence to Richard H. Scheller Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford, CA 94305-5428. Tel.: (650) 723-9075. Fax: (650) 725-4436. E-mail: scheller{at}cmgm.stanford.edu

J.C. Hay's present address is Department of Biology, University of Michigan, 830 North University, Room 3065C, Ann Arbor, MI 48109-1048.


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