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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01269

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Differential effects of a GTP-restricted mutant of Sar1p on segregation of cargo during export from the endoplasmic reticulum

¹ Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK
² Cell Biology and Cell Biophysics Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, 69117, Germany

View larger version (113K): [in a new window]   Fig. 1. Inhibition of transport at 15°C. Cells were transfected with plasmid DNA encoding cargo proteins and incubated at 15°C for 2 hours prior to imaging. ts045-G-CFP (A) colocalises with lumYFP (B) (arrowheads) whereas procollagen-CFP (D) is localised to different discrete structures when compared with lumYFP (arrows) (E). GPI-CFP (G and J) colocalises with both ts045-G-YFP (H) and lumYFP (K). C,F,I,L are merged images of the respective images in the same row. Bars, 10 µm.

View larger version (102K): [in a new window]   Fig. 2. Colocalisation of lumFP, COPII and ERGIC-53 at 15°C. Cells were transfected with plasmid DNA encoding lumYFP and incubated at 15°C for 2 hours prior to fixation, processing for immunofluorescence and imaging. lumYFP (A,D) colocalises with COPII (Sec13p) (B) and ERGIC-53 (E) (arrowheads). C and F are merged images of the respective images in the same row. Bars, 10 µm.

View larger version (92K): [in a new window]   Fig. 3. General dependence of secretory cargo exit on functional Rab proteins and dynactin complex. Cells were co-injected with plasmid DNA encoding either lumYFP (A,C,E) or GPI-YFP (B,D,F) and CFP (A,B), Rab-GDIß (C,D) or p50dynamitin (E,F). LumYFP and GPI-YFP plasmids were injected at a concentration of 10 ng µl–1 with inhibitor encoding plasmids at a concentration of 50 ng µl–1. Panels show the localisation of lumFP or GPI-FP in living cells, imaged at 37°C after 3 hours of expression. Arrowhead in D shows nuclear envelope. Bars, 10 µm.

View larger version (81K): [in a new window]   Fig. 4. Export of GPI-FP from the ER requires GTP hydrolysis by the Sar1p GTPase. (A,B) Cells co-injected with plasmids encoding Sar1p(H79G) and either lumFP (A) or GPI-FP (B). Arrowhead shows localization to the nuclear envelope. (C,D) Cells expressing lumFP (C) or GPI-FP (D) were incubated in BFA for 2 hours. Subsequently, cells were microinjected with GTP--S, and BFA was washed-out for 1 hour (E,F). LumFP exits the ER in the absence of GTP hydrolysis by Sar1p (compare A and E) but GPI-FP remains exclusively within the ER (compare B and F). In addition to clear labelling of the ER, lumFP showed accumulation into punctate structures distributed throughout the cells but concentrated in the juxtanuclear region (C, enlargement, arrowheads). Bars, 10 µm.

View larger version (73K): [in a new window]   Fig. 5. GPI-FP transiently localises to ER export sites but is not incorporated into nascent pre-budding complexes. (A,B) Cells were microinjected with plasmids encoding GPI-FP with Sar1p(H79G), incubated at 37°C in the presence of 5 µg ml–1 BFA for 2 hours and subsequently washed in growth medium and incubated at 37°C for 1 hour (A), or 5 minutes (B) before imaging of living cells. Inset to panel B shows deliberately enhanced contrast to clearly show punctate structures (arrowheads). (C-F) Cells injected with plasmids encoding YFP-Sec23Ap, GPI-CFP and Sar1p(H79G) and incubated in the presence of brefeldin A for 2 hours; cells were then washed and imaged at 37°C. (E,F) Enlarged areas of C and D. Thirty minutes after BFA wash-out, YFP-Sec23Ap (C, enlarged in E) is localised to tubules (arrowheads) that are decorated with punctate structures (arrows). Other punctate structures are clustered around, the perinuclear area. By contrast, GPI-CFP (D) (enlarged in F) remains exclusively localised within the ER (arrow highlights nuclear membrane staining). (G,H) In cells expressing Sar1p(H79G), BFA wash-out results in accumulation of YFP-Sec23Ap (G) in punctate structures clustered in the perinuclear area (arrowheads) that also contain ts045-G-FP (H) (arrowheads). Bars, 5 µm.

View larger version (60K): [in a new window]   Fig. 6. Diffusion-mobility of ts045-G-FP in pre-budding complexes and GPI-FP in the ER membrane. (A) Cells expressing ts045-G and Sar1p(H79G) were imaged before (pre-bleach) and after photobleaching of the central boxed area. Cells were imaged for five frames before bleaching a central region of the cell and monitoring recovery by measuring fluorescence intensity (arbitrary units) within the bleached area (the first post-bleach frame is defined as time zero on the x-axis). Frames were 0.677 seconds apart; bleaching was performed using four scans of a central region of interest with maximum laser power. The first post-bleach image was acquired approximately 4 seconds after the end of the bleaching step. (B) Cells expressing ts045-G-GFP and Sar1p(H79G) at 32°C (), GPI-GFP and Sar1p(H79G) at 32°C (), ts045-G-GFP at 39.5°C (), or ts045-G-GFP at 32°C () were analysed using fluorescence recovery after photobleaching. Data shown is the average from six cells of three independent experiments. Error bars showthe standard error of the mean (s.e.m.). (C) Cells expressing ts045-G-FP were temperature-shifted from 39.5°C to 32°C for 5 minutes and imaged by using FRAP. The cell was bleached within the boxed region and 18 seconds later imaged every 1.3 seconds. The top right hand corner of each panel shows a 2x zoom of the bleached region. Arrowheads with asterisks highlight structures that do not show fluorescence recovery, arrowhead shows a structure that shows limited recover. Arrows highlight structures that move into the bleached region during the post-bleach period. (D) Cells expressing lumFP and Sar1p(H79G) were sequentially imaged at low laser intensity and high laser intensity (FLIP) (within the boxed region only). Fluorescence loss occurs outside the bleached region. In control cells, in which a bleach region was selected away from any cells, no significant loss of fluorescence occurs.

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