FIGURE 1.

Ricin B chain disappears in transfected and transgenic tobacco protoplasts. A, protoplasts transfected with pDHA vector alone (vector) or plasmid encoding ER-targeted ricin B chain (RTB) were radiolabeled with ³⁵S-amino-acids for 1 h and chased with unlabeled amino acids for the times indicated. RTB was immunoprecipitated from separated cell and medium homogenates and analyzed by SDS-PAGE and fluorography. B, protoplasts were treated as in A, but using leaf protoplasts prepared from transgenic tobacco plants constitutively expressing RTB. C, quantitation of the proportion of RTB remaining within cells, secreted into the medium, or unaccounted for after a 5-h chase in transiently expressing or transgenic protoplasts. Mean values are from six or three independent experiments, respectively. Error bars indicate standard deviation. D, protoplasts were transfected with plasmids encoding RTB and pulse-chased as in A. Where indicated, RTB immunoprecipitates were treated for 1 h in the absence or presence of PNGaseF, or protoplasts were preincubated for 1 h with 5 mm DMM before radiolabeling. E, left panel, immunoblot following incubation of medium from vector-transfected protoplasts with buffer (-) or with 150 ng of castor bean purified RTB (+). Right panel, protoplasts expressing vector or RTB were homogenized and incubated with (+) or without (-) anti-RTB antiserum and protein A-Sepharose before the unbound, nonimmunoreactive proteins were resolved by SDS-PAGE and immunoblotted for any nonimmunoprecipitable B chain. In all panels, numbers on the margins of gels indicate molecular mass markers in kilodaltons.