Figure 2.

VSR1, VSR3, and VSR4 are involved in the vacuolar trafficking of soluble cargo proteins. A to C, Western-blot analysis of AALP:GFP, CPY:GFP, or phaseolin trafficking in protoplasts from vsr1vsr3, vsr1vsr4, and vsr5vsr6 double mutant plants. Protoplasts derived from wild-type and indicated vsr mutant plants were transformed AALP:GFP (A), CPY:GFP (B) or phaseolin (C). After 24 h of incubation, protein extracts were prepared from the protoplasts (P) and from the incubation medium (S). Twenty-five percent of protein extracts from the protoplasts and incubation medium were used for western-blot analysis. Targeting efficiency was expressed as a relative value of the amount of precursor (Pre), processed (Pro), and secreted protein (S) to the amount of total protein. Actin was detected as a control for protein leakage from protoplasts and as a loading control. Three independent transformation experiments were performed to obtain an average targeting efficiency. Error bars indicate sd (n = 3). 1/3, vsr1vsr3 mutant; 1/4, vsr1vsr4 mutant; 5/6, vsr5vsr6 mutant. D, Western-blot analysis of invertase:GFP in the wild type and vsr double mutants. Protoplasts were transformed with invertase:GFP and protein extracts were prepared from the protoplasts (P) and from the incubation medium (S). Twenty-five percent of protein extracts from the protoplasts and incubation medium were used for western-blot analysis. [See online article for color version of this figure.]