Efficient prenylation by a plant geranylgeranyl-transferase-I requires a functional CaaL box motif and a proximal polybasic domain

Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common a-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. S equence 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 us ed for biochemical and molecular analysis. The recombinant AtGGT-I prenylat ed efficiently CaaL box fusion proteins in which the a(2) position was occu pied 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 re action rate, and increased the affinity of the enzyme to the protein substr ate by an order of magnitude. GGT-I retained high activity in a temperature range between 24 degreesC and 42 degreesC, and showed increased activity r ate at relatively basic pH values of 7.9 and 8.5. Reverse transcriptase-pol ymerase chain reaction, protein immuno-blots, and transient expression assa ys of green fluorescent protein fusion proteins show that GGT-IB is ubiquit ously expressed in a number of tissues, and that expression levels and prot ein activity were not changed in mutant plants lacking FTase beta -subunit.