Abstract
BackgroundProtein 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.ResultsWe 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 Km but different kcat values in the presence of PFT and PGGT1, indicating that substrate specificities are determined primarily by reactivity rather than binding affinity.ConclusionsThe 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.
Highlights
Protein prenylation is a common post-translational modification in metazoans, protozoans, fungi, and plants
Given this heterogeneity in the specificity of protein farnesyltransferase (PFT) and protein geranylgeranyltransferase type 1 (PGGT1) for certain CaaX proteins, deconvoluting the complex roles of protein prenylation in negative regulation of abscisic acid (ABA) signaling, meristem development, and other fundamental processes poses a significant challenge. To address this problem, we characterized Arabidopsis PFT and PGGT1 with respect to substrate specificity and catalysis. These studies were aimed at answering the following questions: 1) What distinguishes plant CaaX prenyltransferases from animal and fungal prenyltransferases and what gives them their unique substrate specificities? 2) Do the substrate specificities of Arabidopsis PFT and PGGT1 potentially explain the phenotypes of era1 and ggb mutants? The results reported here indicate that Arabidopsis PFT exhibits less specificity for the terminal position of the CaaX motif than PFT enzymes from metazoans and yeast and Arabidopsis PGGT1 exhibits greater specificity for CaaX motifs with leucine in the terminal position than PGGT1 enzymes from metazoans and yeast
Recombinant Arabidopsis PFT is more specific for isoprenoid substrates than PGGT1, whereas PGGT1 is more specific for CaaX substrates To functionally characterize Arabidopsis PFT and PGGT1, we co-expressed the PLP and ERA1 coding sequences in E. coli using the pETDuet-1 vector (PLP was expressed with an amino terminal FLAG tag and ERA1 was expressed with an amino terminal 6 × His tag)
Summary
Protein prenylation is a common post-translational modification in metazoans, protozoans, fungi, and plants. Plants with defects in the GGB gene exhibit increased ABA-induced stomatal closure and auxininduced lateral root formation [12], but without significant developmental phenotypes These observations suggest that at least one geranylgeranylated protein functions as a negative regulator of ABA signaling and at least one functions as a negative regulator of auxin signaling. Prenylated proteins have been implicated in a plethora of other processes, including calcium signal transduction [26,27], response to heat and heavy metal stress [28,29,30], cytokinin biosynthesis [31], and regulation of the cell division cycle [6,32,33] Given these multiple roles, it is surprising that, unlike other organisms, Arabidopsis plants survive without the shared α-subunit of PFT and PGGT1 [14]
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