Fig. 8.

A proportion of zeolin–Nef enters secretory traffic resulting in the production of phaseolin vacuolar fragments. Protoplasts isolated from young leaves of transgenic tobacco expressing zeolin–Nef were subjected to pulse-labelling with [³⁵S]Met and [³⁵S]Cys for 1 h followed by chase for the indicated times. Protoplasts were homogenated with reducing buffer. (A) Proteins were immunoprecipitated with anti-phaseolin, anti-γ-zein, or anti-Nef antibodies, as indicated on top of the panel. (B) Pulse–chase was performed in the presence (+) or absence (–) of BFA; immunoprecipitation was with anti-phaseolin antiserum. (C) Shorter exposure of the fluorograph in (B), showing only the region containing intact zeolin–Nef. In all panels, analysis was by SDS–PAGE and fluorography. The positions of zeolin–Nef (arrowhead) and phaseolin fragmentation products (vertical bar) are marked on the left. Numbers on the right indicate the positions of molecular mass markers, in kilodaltons.