Abstract
In a preceding study 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 there 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, in this initial study 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 now 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
IntroductionCertain 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
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
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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