The plastidial 2-C-methyl-D-erythritol 4-phosphate pathway provides the isoprenyl moiety for protein geranylgeranylation in tobacco BY-2 cells.
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-trans-geranylgeraniol (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-d-erythritol 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.
Gerber
Esther
E
Institut de Biologie Moléculaire des Plantes (Centre National de la Recherche Scientifique, Unité Propre de Recherche 2357, associated with the Université Louis Pasteur), F-67083 Strasbourg, France.
Hemmerlin
Andréa
A
Hartmann
Michael
M
Heintz
Dimitri
D
Hartmann
Marie-Andrée
MA
Mutterer
Jérôme
J
Rodríguez-Concepción
Manuel
M
Boronat
Albert
A
Van Dorsselaer
Alain
A
Rohmer
Michel
M
Crowell
Dring N
DN
Bach
Thomas J
TJ
D002478:Cells, Cultured; D003001:Cloning, Molecular; D004896:Erythritol / Q000031:analogs & derivatives* / Q000378:metabolism; D005578:Fosfomycin / Q000031:analogs & derivatives / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis; D017368:Protein Prenylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism*
C114232:2-C-methylerythritol 4-phosphate; D010940:Plant Proteins; D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; D013403:Sugar Phosphates; D049452:Green Fluorescent Proteins; D005578:Fosfomycin; C024640:fosmidomycin; C002963:geranylgeranyl pyrophosphate; D004896:Erythritol