The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells  

Abstract

Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-transgeranylgeraniol (GGol). Furthermore, 1-deoxy-D-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-D-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-Derythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

S-Carvone Suppresses Cellulase-Induced Capsidiol Production in Nicotiana tabacum by Interfering with Protein Isoprenylation

“…Some proteins are posttranslationally modified with an MEP-derived geranylgeranyl diphosphate that originates from plastids (Gerber et al, 2009). Monoterpenes are often contained in natural essential oils and some of them are known to block the activity of animal and human type I protein prenyltransferases (Crowell et al, 1991;Gelb et al, 1995;Hardcastle et al, 1999).…”

“…Furthermore, efficient incorporation of these inhibitors into leaves cannot be considered certain and needs to be attested. We tested two inhibitors, GGti-2133 that was previously identified as an efficient inhibitor of protein geranylgeranylation in N. tabacum BY-2 cells (Gerber et al, 2009), and FPT-inhibitor I described as specifically inhibiting protein farnesylation. GGti-2133 was found to prevent capsidiol N. tabacum production in elicited leaf discs, but the FPT-inhibitor I did not (Supplemental Table S4).…”

“…To definitively resolve the issue of whether S-carvone blocks protein isoprenylation in plants, an in vivo visualization system for protein geranylgeranylation was utilized (Hartmann et al, 2013). This system is based on transgenic N. tabacum BY-2 cell lines expressing GFP-BD-CVIL bearing a basic domain (BD) and a prenylation motif (CVIL) from rice CaM61 (Gerber et al, 2009). When modified with a geranylgeranyl group, this chimeric protein becomes membrane localized.…”

“…We were not able to detect PPTase activity in cell-free extracts isolated from N. tabacum leaves. For that reason, we tested it in N. tabacum BY-2 cell extracts, in which activity can easily be estimated (Randall et al, 1993;Gerber et al, 2009). A protein extract isolated from a 24-h-old cell suspension was used as an enzyme source.…”

Farnesol kinase is involved in farnesol metabolism, ABA signaling and flower development in Arabidopsis

“…Despite considerable exchange of isoprenoid intermediates between these pathways, the former is primarily responsible for the biosynthesis of sesquiterpene (C 15 ) and triterpene (C 30 ) compounds, including sterols and the farnesyl moiety of farnesylated proteins, whereas the latter is primarily responsible for the biosynthesis of monoterpene (C 10 ), diterpene (C 20 ) and tetraterpene (C 40 ) compounds, including carotenoid pigments and the geranylgeranyl moiety of geranylgeranylated proteins (Randall et al, 1993;Lichtenthaler et al, 1997;Disch et al, 1998;Kasahara et al, 2002;Hemmerlin et al, 2003;Laule et al, 2003;Suzuki and Muranaka, 2007;Gerber et al, 2009). Gibberellins and abscisic acid (ABA) are also products of the plastidial MEP pathway.…”