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. 2001 Aug;126(4):1416-29.
doi: 10.1104/pp.126.4.1416.

Efficient Prenylation by a Plant geranylgeranyltransferase-I Requires a Functional CaaL Box Motif and a Proximal Polybasic Domain

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Efficient Prenylation by a Plant geranylgeranyltransferase-I Requires a Functional CaaL Box Motif and a Proximal Polybasic Domain

D Caldelari et al. Plant Physiol. .
Free PMC article

Abstract

Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common alpha-subunit with farnesyltransferase (FTase) and has a distinct beta-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I beta-subunit (AtGGT-IB) was achieved by a yeast (Saccharomyces cerevisiae) two-hybrid screen, using the tomato (Lycopersicon esculentum) FTase alpha-subunit (FTA) as bait. Sequence and structure analysis revealed that the core active site of GGT-I and FTase are very similar. AtGGT-IA/FTA and AtGGT-IB were co-expressed in baculovirus-infected insect cells to obtain recombinant protein that was used for biochemical and molecular analysis. The recombinant AtGGT-I prenylated efficiently CaaL box fusion proteins in which the a(2) position was occupied by an aliphatic residue, whereas charged or polar residues at the same position greatly reduced the efficiency of prenylation. A polybasic domain proximal to the CaaL box motif induced a 5-fold increase in the maximal reaction rate, and increased the affinity of the enzyme to the protein substrate by an order of magnitude. GGT-I retained high activity in a temperature range between 24 degrees C and 42 degrees C, and showed increased activity rate at relatively basic pH values of 7.9 and 8.5. Reverse transcriptase-polymerase chain reaction, protein immuno-blots, and transient expression assays of green fluorescent protein fusion proteins show that GGT-IB is ubiquitously expressed in a number of tissues, and that expression levels and protein activity were not changed in mutant plants lacking FTase beta-subunit.

Figures

Figure 1
Figure 1
Amino acid sequence alignment of protein geranylgeranyltransferase β-subunits and the Arabidopsis FTB. Sequence alignments were established by using the John Hein method (Lasergene). Amino acid identities between the Arabidopsis AtGGT-IB and AtFTB proteins, and GGT-I β-subunits from yeast and human, are indicated by black boxes. Dashes denote gaps formed by the alignment algorithm. In some regions the alignment program failed to align the AtFTB sequence correctly because of the divergence from the yeast sequence. Molecular functions were assigned to some of the residues using the crystal structure of the rat FTase as a model. Green A denotes residues in the hydrophobic pocket, blue P denotes residues that interact with the CaaX peptide, brown PP denotes residues that interact with the diphosphate group of FPP (or GGPP), red Z denotes the ligands of the catalytic zinc atom, and blue arrows denote residues that are conserved between all GGT-I but differ in FTBs. AtGGT-IB was isolated as a 1,351-bp clone with 1,128-bp open reading frame. Low stringency DNA-blot analysis indicates that AtGGT-IB exists in a single copy form in the Arabidopsis genome (data not shown). RNA-blot analysis (data not shown) and comparison to the genomic sequence of AtGGT-IB (GenBank accession no. ATAC004218) indicate that AtGGT-IB represents a full-length clone. The GenBank accession nos. for the aligned sequences are as follows: AtGGT-IB, AF311225; human GGT-IB, L25441; yeast (S. cerevisiae) GGT-IB, M74109; and Arabidopsis AtFTB, AF214106.
Figure 2
Figure 2
Expression patterns of AtGGT-IB RNA and protein. A, Dot-blot analysis of GGT-IB, Ubiquitin, and APETALA1 RT-PCR products prepared from wt Col-0 and era1-2 Arabidopsis plants. Fl, Flower; Le, leaf; St, stem; Ro, root. The RT-PCR was carried out as described in “Materials and Methods” B, Immunoblot analysis of AtGGT-IB expression in wt Col-0 and era1-2 Arabidopsis plants using α-AtGGT-IB polyclonal Abs (“Materials and Methods”). Fl, Flowers; Le, leaf; St, stem; Si, silique; C, recombinant GGT-IB control. The control protein appears larger because it contained additional residues that originated from the pFastBacHTa cloning vector (“Materials and Methods”).
Figure 3
Figure 3
Recombinant FTase and GGT-I proteins. Stained gel showing recombinant (His)6-tagged FTase (FT) and GGT-I that were expressed in baculovirus-infected insect cells, and purified over an Ni-NTA column (“Materials and Methods”). The Mr difference between FTB and GGT-I β-subunit (GGB) is visible. FT/GGA, The common α-subunit.
Figure 4
Figure 4
Prenylation efficiency of different gluthatione-S-transferase (GST)-CaaL box fusion proteins. Prenylation reactions were carried out with GST alone or with GST-CaaL fusion proteins, as indicated on the figure, with either GGPP (G) or FPP (F) as prenyl group donors. Reactions were terminated by denaturing proteins and separating them on SDS gels, which were in turn fluorographed and exposed to x-ray film (A). Bands in A were quantified and the band with the maximum intensity was given the value of 1 (B). Cyclophilin (CYP), CYP from Solanum commersonii (wild potato), GenBank accession no. U92087; AUX22-1 auxin-induced protein from Arabidopsis, GenBank accession no. L15450; MsMIP, membrane channel protein from alfalfa (Medicago sativa), EMBL accession no. L36881; CaM53 Ca2+, calmodulin from petunia, GenBank accession no. M80831.
Figure 5
Figure 5
CaM53, GST-BDCaM53, and GST-CTIL protein substrates and their prenylation. A, Stained SDS-PAGE of the purified protein substrates. B, Fluorogram showing prenylation of GST-BDCaM53 and GST-CTIL fusion recombinant protein. C, Fluorogram showing prenylation of CaM53 and GST-CTIL recombinant proteins. Numbers in A through C denote molecular mass in kilodaltons. D, Schematic presentation of CaM53, GST-BDCaM53, and GST-CTIL. BD, Basic domain; EF, Ca2+-binding EF hands.
Figure 6
Figure 6
Substrate saturation curves for prenylation of CaM53 and GST-CTIL by GGT-I. Prenylation reactions were carried out with concentrations of CaM53 and GST-CTIL as indicated on the figure. All other conditions were as described in “Materials and Methods.” Reactions were terminated by separating the protein products on SDS gels, which were, in turn, fluorographed and exposed to x-ray films (A and C). The fluorograms in A were exposed for 60 h, and the fluorogram in C for 48 h. C, −, Denotes reactions carried out with boiled GGT-I enzyme; +, reactions carried out with active GGT-I. B and D, Quantification of the bands on fluorograms in A and C, respectively. Values for each band pair were averaged, and the maximal intensity was given a value of 1. The kinetics of the prenylation of CaM53 appeared more accurate following a shorter (48 h) exposure period of the film (compare A and B with C and D). However, the radiolabeled GST-CTIL was barely detectable when films were exposed for less then 60 h (data not shown).
Figure 7
Figure 7
Substrate saturation curves for prenylation of GST-BDCaM53 and GST-CTIL by GGT-I. Prenylation reactions were carried out with concentrations of GST-BDCaM53 and GST-CTIL as indicated in the figure. All other conditions were as described in “Materials and Methods.” Reactions were carried out in triplicates, and were terminated by separating between protein-incorporated and free GGPP using the acidic ethanol method (“Materials and Methods”). Each graph point represents the average value of each of the three reactions and bars are sd values. To express the incorporation of GGPP in values of pmol min−1, the radioactivity of known amount of [3H]GGPP was measured by scintillation counting.
Figure 8
Figure 8
How does GGT-I activity depend on temperature? Prenylation reactions of CaM53 were carried out at different temperatures, as indicated on the figure. All other conditions were as described (“Materials and Methods”). Separation of the protein products on SDS gels terminated reactions, followed by fluorography of gels and exposure to x-ray films (A). Values for each band pair were averaged, and the maximal intensity was given a value of 1. −, Denotes reactions carried out with boiled GGT-I enzyme; +, reactions carried out with active GGT-I.
Figure 9
Figure 9
Regulation of GGT-I by pH. Prenylation reactions of GST-BDCaM53 were carried out at different pH values as indicated on the figure. All other conditions were as described (“Materials and Methods”). Reactions were carried out in triplicates, and were terminated by separating between protein-incorporated and free GGPP using the acidic ethanol method (“Materials and Methods”). Each graph point represents the average value of each of the three reactions and bars are sd values. The incorporation of GGPP into substrate protein was calculated as described in Figure 7.
Figure 10
Figure 10
The activity of GGT-I in era1-2 mutants. A GFP-BDCaM53 fusion protein was transiently expressed in leaves of wt Col-0 and era1-2 mutants following transformation by biolistic bombardments. The intracellular localization of the fusion protein was determined by imaging cell with confocal microscope. Prenylated GFP-BDCaM53 protein was located to the plasma membrane resulting in green fluorescence at the periphery of the cells. Un-prenylated fusion protein accumulated in the nucleus (N; Rodríguez-Concepción et al., 1999b). The red fluorescence came from plastids (P). Bars are 20 microns.

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