Fig. 2. The PCP leader contains a signal peptide that targets a reporter gene to canine microsomes in vitro. (A) A schematic view of the G. polyedra PCP leader used (top) shows two hydrophobic regions (numbered black boxes) separated by an S/T-rich region (white box) fused to luciferase (line). The small arrow indicates the potential AXA signal peptidase site. This construct was transcribed and translated in vitro in rabbit reticulocyte lysates (RRL) with the additions as illustrated. A smaller protein is produced after translation in the presence of canine microsomes, confirming cleavage after the first hydrophobic region. The topology of the protein, deduced from its susceptibility to trypsin digestion, is shown schematically on the right (small arrow indicates cleavage by the signal peptidase inside the vesicle). Note that the bulk of the protein remains outside the microsomal membrane. (B) A schematic view of the luciferase reporter fused with a modified G. polyedra PCP leader (top) lacking the first hydrophobic region. This construct was also translated as above. Most of the translation product is found in the pellet (P) rather than the supernatant (S) after ultracentrifugation, suggesting that it may be inserted into the microsomal membrane as found in Euglena. The predicted topology is again shown schematically on the right.