Figure 4.

Faithful targeting of a membrane protein and a cytosolic reporter protein expressed from a polyprotein construct. a, The cytosolic reporter protein, GUS, and a plant membrane protein, F5H, were separated by FMDV 2A and encoded as a polyprotein with a single ORF. The arrow indicates the position where the polyprotein would be expected to dissociate, on translation, at the carboxy terminus of 2A. b, Products obtained when the polyprotein construct was translated by TNT reticulocyte lysate in vitro in the absence (−) and presence (+) of microsome membranes. The left-hand lanes show total translation products. The right-hand lanes show the products present in the supernatant and in pelleted membranes after fractionation, by ultracentrifugation through a Suc cushion, of a translation reaction performed in the presence of microsomes. c, Protein extracts from plants transformed with pGUS2A-F5H and wild-type plants were assayed for GUS activity. Selected wild-type and transgenic plants were subjected to Klason lignin analysis to determine the effect of F5H expression on lignin content. Triplicate lignin measurements were made for each plant, and the mean Klason lignin is shown [as a % of extractive-free cell wall residue (CWR)]. Individual assay values varied from the mean by less than 2% (maximum sd = ±0.33).