Figure 6.

Secretion of the 45-kD Processed Form of Zeolin.

(A) All protoplasts were prepared from transgenic tobacco expressing zeolin. Protoplasts were pulse-labeled with [³⁵S]Met and [³⁵S]Cys for 1 h followed by an 8-h chase; the incubation medium was collected and divided into two equal aliquots: one was homogenated and immunoselected with anti-phaseolin antiserum (lane 1); the other was adjusted to 20 mM 2-ME and added to unlabeled protoplasts that had been pretreated for 8 h with 20 mM 2-ME, and after an additional 16 h of incubation, the medium was homogenated and immunoselected with anti-phaseolin antiserum (lane 2). As a control, the medium from protoplasts labeled for 1 h and subjected to a 24-h chase in the presence of 20 mM 2-ME was also immunoselected (lane 3).

(B) Protoplasts from transgenic tobacco expressing zeolin were preincubated for 45 min in the presence of BFA and maintained in the presence of the drug for the whole pulse–chase. Pulse with [³⁵S]Met and [³⁵S]Cys was for 1 h, and chase was for the indicated times, in the presence of 20 mM 2-ME. Total homogenates were prepared from protoplasts or incubation medium, using homogenation buffer supplemented with 2-ME (medium) or without the reducing agent (protoplasts), and immunoselected using anti-phaseolin antiserum.

Analysis was by SDS-PAGE and fluorography. The positions of zeolin (arrowheads), the 45-kD form (arrow), and the 95-kD putative O-glycosylated zeolin (asterisk) are indicated. Numbers at left indicate the positions of molecular mass markers in kilodaltons.