title: Sar1 localizes at the rims of COPII-coated membranes in vivo.
abstract: The Sar1 GTPase controls coat assembly on coat protein complex II (COPII)-coated vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi. The GTP-bound form of Sar1, activated by the ER-localized guanine nucleotide exchange factor (GEF) Sec12, associates with the ER membrane. GTP hydrolysis by Sar1, stimulated by the COPII-vesicle-localized GTPase-activating protein (GAP) Sec23, in turn causes Sar1 to dissociate from the membrane. Thus, Sar1 is cycled between active and inactive states, and on and off vesicle membranes, 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) in a manner that was dependent 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 the reversible membrane association of Sar1 GTPase leads to its localization being restricted to the rims of COPII-coated membranes in vivo.
authors.affiliation: Live Cell Super-Resolution Imaging Research Team, RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan kkurokawa@riken.jp.
authors.forename: Kazuo
authors.identifier: 0000-0003-3549-4795
authors.initials: K
authors.lastname: Kurokawa
authors.affiliation: Live Cell Super-Resolution Imaging Research Team, RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Laboratory of Molecular Cell Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Suda
authors.affiliation: Live Cell Super-Resolution Imaging Research Team, RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakano
mesh_terms: D022181:COP-Coated Vesicles / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D006160:Guanosine Triphosphate; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae
keywords: COPII-coated vesicle; ER; GTP hydrolysis; Sar1; Sec16