The CaaX specificities of Arabidopsis protein prenyltransferases explain era1 and ggb phenotypes

Abstract

Background: Protein prenylation is a common post-translational modification in metazoans, protozoans, fungi, and plants. This modification, which mediates protein-membrane and protein-protein interactions, is characterized by the covalent attachment of a fifteen-carbon farnesyl or twenty-carbon geranylgeranyl group to the cysteine residue of a carboxyl terminal CaaX motif. In Arabidopsis, era1 mutants lacking protein farnesyltransferase exhibit enlarged meristems, supernumerary floral organs, an enhanced response to abscisic acid (ABA), and drought tolerance. In contrast, ggb mutants lacking protein geranylgeranyltransferase type 1 exhibit subtle changes in ABA and auxin responsiveness, but develop normally.

Results: We have expressed recombinant Arabidopsis protein farnesyltransferase (PFT) and protein geranylgeranyltransferase type 1 (PGGT1) in E. coli and characterized purified enzymes with respect to kinetic constants and substrate specificities. Our results indicate that, whereas PFT exhibits little specificity for the terminal amino acid of the CaaX motif, PGGT1 exclusively prenylates CaaX proteins with a leucine in the terminal position. Moreover, we found that different substrates exhibit similar K(m) but different k(cat) values in the presence of PFT and PGGT1, indicating that substrate specificities are determined primarily by reactivity rather than binding affinity.

Conclusions: The data presented here potentially explain the relatively strong phenotype of era1 mutants and weak phenotype of ggb mutants. Specifically, the substrate specificities of PFT and PGGT1 suggest that PFT can compensate for loss of PGGT1 in ggb mutants more effectively than PGGT1 can compensate for loss of PFT in era1 mutants. Moreover, our results indicate that PFT and PGGT1 substrate specificities are primarily due to differences in catalysis, rather than differences in substrate binding.

Figure 1

Protein prenylation and processing in eukaryotes. A, prenylation and processing of farnesylated CaaX proteins. B, prenylation and processing of geranylgeranylated CaaX proteins. C, prenylation of RAB GTPases.

Figure 2

Substrate specificity of recombinant Arabidopsis PFT. Quantitative filter assay data are shown for recombinant Arabidopsis PFT in the presence of [1-³H]FPP, [1-³H]GGPP, and 32 distinct GFP-BD-CaaX substrates. The CaaX substrates are grouped with the variable amino acid indicated in red. Asterisks indicate PFT-catalyzed protein geranylgeranylation detectable after 4 days by SDS-PAGE and fluorography. The red line indicates background. The standard error of the mean is shown.

Figure 3

Radiofluorograms of prenylation assays performed in the presence of recombinant Arabidopsis PFT. Radiofluorograms corresponding to the quantitative filter assay data in Figure 2 are shown.

Figure 4

Substrate specificity of recombinant Arabidopsis PGGT I. Quantitative filter assay data are shown for recombinant Arabidopsis PGGT I in the presence of [1-³H]FPP, [1-³H]GGPP, and 32 distinct CaaX substrates. The CaaX substrates are grouped with the variable amino acid indicated in red. Asterisks indicate PGGT1-catalyzed protein farnesylation detectable after 4 days by SDS-PAGE and fluorography. The red line indicates background. The standard error of the mean is shown.

Figure 5

Radiofluorograms of prenylation assays performed in the presence of recombinant Arabidopsis PGGT1. Radiofluorograms corresponding to the quantitative filter assay data in Figure 4 are shown.

Figure 6

Activity and substrate specificity of IMAC-purified recombinant Arabidopsis PFT and PGGT I. Left Panel: E. coli extracts before Co²⁺-IMAC, unbound proteins, and bound (IMAC-purified) proteins are shown. An E. coli protein at 42 kDa, which co-migrates with the PGGT I β-subunit is present at low levels in the purified PFT and PGGT I samples. Right Panel: quantitative activity data for purified PFT and PGGT I. The standard error of the mean is shown.

Figure 7

Lineweaver-Burk plots for purified PFT. A, GFP-BD-CVIM and [1-³H]FPP were used as substrates. B, GFP-BD-CVIQ and [1-³H]FPP were used as substrates. C, GFP-BD-CVII and [1-³H]FPP were used as substrates. D, GFP-BD-CVIL and [1-³H]FPP were used as substrates. The standard error of the mean is shown.

Figure 8

Lineweaver-Burk plots for purified PGGT1. A, GFP-BD-CVIL and [1-³H]GGPP were used as substrates. B, GFP-BD-CVII and [1-³H]GGPP were used as substrates. C, GFP-BD-CVIQ and [1-³H]GGPP were used as substrates. D, GFP-BD-CVIM and [1-³H]GGPP were used as substrates. The standard error of the mean is shown.