Figure 4
From: Experimental detection of proteolytic activity in a signal peptide peptidase of Arabidopsis thaliana
Expression of the SPP-GFP fusion protein in yeast. (A) Soluble and membrane fractions were analyzed by 12% SDS-PAGE using in-gel fluorescence. The protein concentrations loaded were as follows: Soluble fractions in the left panel; lane 1, AtSPP-GFP, 117 μg; lane 2, AtSPP-GFP, 340 μg; lane 3, HsSPP-GFP, 56 μg; and lane 4, vector, 56 μg. Membrane fractions in the right panel: lane 5, AtSPP-GFP, 306 μg; lane 6, AtSPP-GFP, 1120 μg; lane 7, HsSPP-GFP, 158 μg; and lane 8, vector, 161 μg. Detection of GFP fluorescence in the gel was carried out by exposure to blue light (EPI) at 460 nm with a cut-off filter. The arrows indicate the location of the GFP fusion proteins. (B) Proteolytic activity of the membrane fraction of GFP-fusion protein. Solubilized membrane fractions of yeast cells overexpressing the vector control (183.1 μg) HsSPP-GFP (197.5 μg) and AtSPP-GFP (55 μg) proteins were incubated for 15 h in the presence or absence of the inhibitor. Lane 1: control (without inhibitor); and lane 2: 50 μM (Z-LL)2-ketone. Arrows indicate the proteolytic products from myc-Prl-PP-FLAG detected with the anti-myc antibody.