Fig. 4.

Zein–Nef does not traffic through the Golgi complex. (A) Proteins were extracted with reducing buffer from young leaves of tobacco plants expressing zein–Nef or from wild-type tobacco (control). After incubation in the presence (+) or absence (–) of endoglycosidase H (Endo H), proteins were analysed by SDS–PAGE and protein blot with anti-Nef antibody. (B) Longer exposure of the blot shown in (A). (C) Protoplasts isolated from plants expressing zein–Nef were treated with brefeldin A and subjected to pulse-labelling with [³⁵S]Met and [³⁵S]Cys for 1 h followed by chase for 0 h or 2 h; protoplasts were homogenated with reducing buffer and immunoprecipitation was performed with anti-Nef antibody. The immunoprecipitates were incubated in the presence (+) or absence (–) of endoglycosidase H and analysed by SDS–PAGE and fluorography. In (A) and (C), the positions of glycosylated (arrowhead) and deglycosylated (dot) zein–Nef are indicated on the right. In each panel, numbers on the left indicate the position of molecular mass markers, in kilodaltons.