FIGURE 3.

Ricin B chain is not stabilized when secretion is blocked. A, protoplasts were transfected with plasmids encoding ER-targeted RTB or phaseolin and subjected to pulse-chase as described under “Experimental Procedures.” Where indicated, protoplasts were preincubated for 1 h with 36 μm BFA before radiolabeling. IPs were analyzed by SDS-PAGE and fluorography. The black arrowhead shows the position of full-length phaseolin, and the vertical bar indicates the vacuolar-generated fragments of phaseolin. B, protoplasts were treated as in A but IPs, from combined cell and medium samples, were treated for 1 h in the presence of PNGaseF prior to SDS-PAGE and fluorography. The single and double asterisks indicate the position of PNGase-resistant singly and doubly glycosylated RTB, respectively. The graph shows densitometric quantifications of RTB made in the presence or absence of BFA, expressed as percentages of the total RTB present at the end of the pulse. Average values from three independent experiments are shown; Error bars indicate standard deviation. C, protoplasts were transfected with constructs encoding RTB or phaseolin and, where indicated, co-transfected with a Sec12 expressing plasmid. Protoplasts were treated as in B. The black arrowhead and vertical bar are as described in A. The asterisks are the same as in B. The graph shows quantification calculated as in B. In all panels, numbers at the margins of gels indicate molecular mass markers in kilodaltons.