Efficient prenylation by a plant geranylgeranyltransferase-I requires a functional Caal box motif and a proximal polybasic domain

D. Caldelari, H. Sternberg, M. Rodríguez-Concepción, W. Gruissem, S. Yalovsky*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common α-subunit with farnesyltransferase (FTase) and has a distinct β-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I β-subunit (AtGGT-IB) was achieved by a yeast (Saccharomyces cerevisiae) two-hybrid screen, using the tomato (Lycopersicon esculentum) FTase α-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 a2 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°C and 42°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 β-subunit.

Original languageEnglish
Pages (from-to)1416-1429
Number of pages14
JournalPlant Physiology
Volume126
Issue number4
DOIs
StatePublished - 2001

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