Fig. 1. Expression-level-dependent and ascorbate-dependent localisation of PC-GFP. Cells expressing low levels of PC-GFP, 3 hours after microinjection of plasmid DNA, localise the protein to the ER in the absence of ascorbate (A), but 10 minutes after the addition of ascorbate, PC-FP localises to punctate TCs that traffic to the Golgi (B). Time lapse movies showing the dynamics of procollagen in the absence (Movie 1) and presence (movie 2) of ascorbate are available at jcs.biologists.com/supplemental . In contrast cells expressing high levels of PC-GFP, 6 hours after microinjection of plasmid DNA, localise the protein, in the absence of ascorbate, to aggregates located within the ER (C). The exposure times for (B) and (C) are the same; arrowheads are ndicated TCs in (B) and aggregates in (C). Cells expressing such high levels of PC-FP were not used during any of the experiments described here. PC-GFP is intact when expressed in cells. Bars=5 µm. (D) shows an immunoblot with the anti-procollagen antibody LF-39 of mock-transfected HeLa cells (lane 1) or HeLa cells transfected with PC-GFP (lane 2). The mobility shift in lane 2 is caused by the presence of the GFP moiety and partial glycosylation.

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