Inhibition and Regulation of Isoprenoid Biosynthetic Pathways in Plants
Author | : Michael Hartmann |
Publisher | : |
Total Pages | : 233 |
Release | : 2010 |
ISBN-10 | : OCLC:868427312 |
ISBN-13 | : |
Rating | : 4/5 (12 Downloads) |
Book excerpt: Higher plants synthesize their isoprenoids through two different routes, the cytosolic mevalonic acid (MVA) pathway and the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. By contrast, many human pathogenes rely exclusively on the MEP pathway for the synthesis of their isoprenoids. As this pathway does not occur in mammals, it represents an attractive target for the design of biopharmaceuticals and herbicides. Our group has recently established an in vivo visualization system for the geranylgeranylation of proteins, on the basis of a stably transformed tobacco BY-2 cell line, expressing a GFP fused to the prenylable, C-terminal basic domain of a rice calmodulin, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). The use of pathway-specific inhibitors revealed that inhibition of the MEP pathway, as well as inhibition of the geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. By contrast, the inhibition of the MVA pathway did not affect the localization. Complementation assays with pathway-specific intermediates further confirmed that the precursors for the prenylation of the fusion protein are predominantly provided by the MEP pathway.To optimize the initial test system, the existing cell line was transformed with an inducible vector, driving the expression of a nuclear marker protein that allows an easier quantification of total cells by image analysis software. Finally, the assay was adapted for the use of 96-well glass-bottom plates and provided us with an inexpensive, visual system to screen for potential inhibitors of the MEP pathway.