Sar1 GTPase controls coat assembly on COPII-coated vesicle, which mediates protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The GTP-bound form of Sar1, activated by ER-localized guanine nucleotide exchange factor (GEF) Sec12, associates with the ER membrane. GTP hydrolysis by Sar1, stimulated by COPII-vesicle-localized GTPase activating protein (GAP) Sec23, in turn dissociates Sar1 from the membrane. Thus Sar1 is cycled between active and inactive and on and off vesicle membrane, but its precise spatiotemporal regulation remains unknown. Here, we examined Sar1 localization on COPII-coated membranes in living Saccharomyces cerevisiae cells. Two-dimensional (2D) observation demonstrated that Sar1 showed modest accumulation around the ER exit sites (ERES) depending on Sec16 function. Detailed three-dimensional (3D) observation further demonstrated that Sar1 localized at the rims of the COPII-coated membranes, but was excluded from the rest of the COPII membranes. Additionally, a GTP-locked form of Sar1 induced abnormally enlarged COPII-coated structures and covered the entirety of these structures. These results suggested that reversible membrane association of Sar1 GTPase restricted its localization to the rims of COPII-coated membranes in vivo.
- Received March 14, 2016.
- Accepted July 12, 2016.
- © 2016. Published by The Company of Biologists Ltd