Abstract
We have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.
Highlights
In eukaryotic cells, certain proteins are modified by a series of posttranslational modifications, leading to the creation of a lipidated, hydrophobic domain at the carboxyl terminus of the protein
This C-terminal CaaX motif is recognized by cytosolic CaaX protein isoprenyl transferases, which either attach a 15-carbon farnesyl unit, a reaction catalyzed by a protein farnesyl transferase (PFT), or a 20-carbon geranylgeranyl unit, a reaction catalyzed by protein geranylgeranyl transferase (PGGT1) to the cysteine of the CaaX motif via a thioether bond
The importance of clonal selection of transformed cell lines According to Carpenter[37,38], in chemical screening based on fluorescence-imaging, intensity might fluctuate from cell to cell for various reasons, including differences in cell cycle position, stochastic variations in gene expression, pre-existing amounts of proteins and metabolites in each cell and micro-environmental differences
Summary
Certain proteins (i.e., members of the Ras superfamily of GTPases in vertebrates) are modified by a series of posttranslational modifications, leading to the creation of a lipidated, hydrophobic domain at the carboxyl terminus of the protein. Protein isoprenylation depends on the presence of a carboxy-terminal tetrapeptide in target proteins, the CaaX motif (‘C’ refers to cysteine, ‘a’ denotes an aliphatic amino acid and ‘X’ represents a specific amino acid)[1,2,3,4] This C-terminal CaaX motif is recognized by cytosolic CaaX protein isoprenyl transferases, which either attach a 15-carbon farnesyl unit (from farnesyl diphosphate, FPP), a reaction catalyzed by a protein farnesyl transferase (PFT), or a 20-carbon geranylgeranyl unit (from geranylgeranyl diphosphate, GGPP), a reaction catalyzed by protein geranylgeranyl transferase (PGGT1) to the cysteine of the CaaX motif via a thioether bond. Both PFT and PGGT1 are heterodimeric enzymes that share a common α-subunit whereas their respective β-subunit is encoded by different genes[3,4,6,9]
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