Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A

Potato virus X (PVX) has been used as an expression vector to target the gr een fluorescent protein (GFP) from the jellyfish Aequorea victoria to the e ndoplasmic reticulum (ER) of tobacco (Nicotiana clevelandii L.) leaves. Exp ression of free GFP resulted in strong cytoplasmic fluorescence with organe lles being imaged in negative contrast. Translocation of GFP into the lumen of the ER was mediated by the use of the sporamin signal peptide. Retentio n of GFP in the ER was facilitated by the splicing of the ER retrieval/rete ntion tetrapeptide, KDEL to the carboxy terminus of GFP. Fluorescence of GF P was restricted to a labile cortical network of ER tubules with occasional small lamellae and to streaming trans-vacuolar strands. Secretion of ER-ta rgeted GFP was inhibited both by cold shock and low concentrations of the s ecretory inhibitor brefeldin A. However, both prolonged cold and prolonged incubation in brefeldin A resulted in the recovery of secretory capability. In leaves infected with the GFP-KDEL construct, high concentrations of bre feldin A induced the tubular network of cortical ER to transform into large lamellae or sheets which reverted to the tubular network on removal of the drug.