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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.

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