Fig. 1.

Localization and transport of GAT1-SSS. (A) Schematic representation of GAT1 showing its 12 transmembrane segments and the C-terminus. The motif replaced by serines in the mutant GAT1-SSS is highlighted by the red box. (B) A HeLa cell expressing YFP-tagged GAT1-SSS. The image was captured 24 hours after transfection by confocal laser scanning microscopy (Leica SPE). (C) HeLa cells were co-transfected with plasmids encoding YFP-tagged GAT1-SSS and CFP-tagged reticulon 2 (CFP-RTN2). After 24 hours cells were fixed and imaged by confocal laser scanning microscopy (Leica SPE). Merged image is on the right. (D) HEK293 cells were transfected with plasmids encoding YFP-tagged GAT1-SSS (upper panels) or YFP-tagged GAT1-RL/AS (lower panels). Arrows indicate the bleached region. FRAP was recorded as described in the Materials and Methods. Fluorescence intensities were expressed as percentage of fluorescence measured prior to bleaching, and the data points were subjected to curvilinear regression using an equation for a monoexponential rise from a basal value to obtain the rate constant for fluorescence recovery and the mobile fraction. Data are means ± s.e.m. from four or five independent experiments. (E) YFP-GAT1-SSS, or GAT1-Δ37, and Sar1a-T39N were transiently co-expressed in HEK293 cells. On the next day, semi-intact cells were prepared and the in vitro budding assay was performed as described in the Materials and Methods. (F,G) Vesicle-budding assay performed in semi-intact cells transfected with Sar1a-T39N. Budding of endogenous ERGIC-53 (F) and CLIMP-63 (G) was determined as in panel E.