Digestion with endoglycosidase H was performed using endo Hf (New England Biolabs; see the manufacturer’s specifications for the compositions of storage, denaturing, and reaction buffers). a process that does not occur in maize endosperm. The secreted polypeptides are immunoselected irrespective of the redox conditions of the homogenation buffer (see Supplemental Figure 2 online), indicating that they are soluble, but they represent a very minor proportion of radioactive zeolin. The effect of 2-ME on the solubility, traffic, and processing of zeolin was next determined. The reducing agent was included in the protoplast incubation medium during the chase but not in the homogenation buffer. 2-ME increased the solubility of intact zeolin during the chase (Figures 4A and ?and4B,4B, cf. zeolin in protoplasts at the different chase points in the presence and absence of 2-ME). In the experiment shown in Figure 4A, the major effect on the pattern of zeolin processing and ST3932 traffic was a marked increase in the secretion of the 45-kD processed form. A polypeptide with Rabbit Polyclonal to RTCD1 the same molecular mass became detectable also intracellularly during the chase, probably representing the intermediate of secretion. In several repetitions of this experiment, we consistently observed a marked increase of zeolin solubility during the chase in the presence of 2-ME. In some experiments, the increase in secretion of the 45-kD form was less marked, whereas the secreted 95-kD form and/or phaseolin vacuolar fragments became more abundant (Figure 4B). Protein blot analysis of protoplasts and incubation medium confirmed the pulseCchase results: after 24 h of treatment with 2-ME, the 45- and 95-kD forms clearly accumulated in the incubation medium (Figure 5, cf. lanes 7 and 8). Open in a separate window Figure 4. 2-ME Enhances the Intracellular Traffic of Zeolin. Protoplasts from transgenic tobacco expressing zeolin were pulse-labeled with [35S]Met and [35S]Cys for 1 h and subjected to chase for the indicated times in the presence (+) or absence (?) 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. (A) and (B) display results from fully independent experiments. The positions of zeolin (arrowheads), the 45-kD form (arrows), phaseolin fragments (vertical bars), and the 95-kD putative shows the N-terminal region, containing the repeated domain, is definitely fundamental for ER retention, whereas the C-terminal region is necessary for assembly into PBs (Geli et al., 1994). Our studies on zeolin show the C-terminal region, which contains the domains homologous with the 2S albumins, is indeed not strictly necessary for PB formation (Mainieri et al., 2004). A synthetic version of the eight VHLPPP repeats of the -zein N-terminal region forms an amphipathic polyproline II structure ST3932 and interacts with liposomes prepared with soybean (cv Petit Havana SR1) or transgenic vegetation expressing zeolin (Mainieri et al., 2004), T343F phaseolin (Pedrazzini et al., 1997), 418 phaseolin (Frigerio et al., 1998), or IgA/G (Ma et al., 1994) were cultured in axenic conditions. Protoplasts were prepared from young (4 to 7 cm long) leaves as explained (Pedrazzini et al., 1994). PulseCchase labeling of protoplasts was performed using Pro-Mix (a mixture of [35S]Met and [35S]Cys; Amersham Biosciences) as explained (Pedrazzini et al., 1997). For treatments with 2-ME (Sigma-Aldrich), the reducing agent was added at the end of pulse-labeling at the appropriate concentrations. For treatment with BFA (Roche), the inhibitor was supplemented to the protoplast incubation medium at 10 g/mL at 45 min before radioactive labeling and was managed ST3932 at the same concentration throughout the pulseCchase. Protoplasts were collected immediately after labeling by the addition of 3 quantities of ice-cold W5 medium (154 mM NaCl, 5 mM KCl, 125 mM CaCl22H2O, and 5 mM glucose) and centrifugation at 60and 4C for 10 min. The supernatant (incubation medium) comprising secreted proteins and the protoplast pellet were freezing in liquid nitrogen and stored at ?80C, but freezing was avoided for subcellular fractionation experiments. For transient protein expression, protoplasts were isolated from small leaves of wild-type tobacco SR1 plants cultivated in axenic conditions and subjected to polyethylene glycolCmediated transfection as explained (Pedrazzini et al., 1997) using 80 g of plasmid. After over night recovery, protoplasts were subjected to pulseCchase labeling as explained above. Protoplast Homogenation, Protein Immunoselection, and Protein Blotting Homogenization of the protoplast incubation medium was performed by adding to frozen samples 2 quantities of ice-cold 1.5 protoplast homogenation buffer (150 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1.5 mM EDTA, 1.5% Triton.