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Plant Physiol, August 2001, Vol. 126, pp. 1416-1429
Efficient Prenylation by a Plant Geranylgeranyltransferase-I
Requires a Functional CaaL Box Motif and a Proximal Polybasic
Domain1
Daniela
Caldelari,2
Hasana
Sternberg,
Manuel
Rodríguez-Concepción,3
Wilhelm
Gruissem,4 and
Shaul
Yalovsky*
Department of Plant and Microbial Biology, University of
California, Berkeley, California 94720-3102 (D.C., M.R.-C.,
W.G.); and Department of Plant Sciences, Tel Aviv University, Ramat
Aviv, Tel Aviv 69978, Israel (H.S., S.Y.)
Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that
shares a common  -subunit with farnesyltransferase (FTase) and has a
distinct  -subunit. GGT-I preferentially modifies proteins, which
terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I
-subunit (AtGGT-IB) was achieved by a yeast
(Saccharomyces cerevisiae) two-hybrid screen, using the
tomato (Lycopersicon esculentum) FTase -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 -subunit.
1
This research was supported by The Israel
Science Foundation (grant no. 571/99 to S.Y.), by the Binational
Science Foundation (grant no. 1999423 to S.Y.), and by the U.S.
Department of Energy (grant no. 85ER13375 to W.G.). D.C. was supported
by a fellowship from the Swiss National Science Foundation, and M.R.C.
had support from the Spanish Ministry of Education and Culture.
2
Present address: Institut d'Écologie, Laboratoire
de Biologie et Physiologie Végétales, Université de
Lausanne, Switzerland.
3
Present address: Department of Biochemistry and
Molecular Biology, University of Barcelona, Spain.
4
Present address: Institute of Plant Sciences, Swiss
Federal Institute of Technology, ETH Zentrum, LFW E57.1, CH-8092
Zurich, Switzerland.
*
Corresponding author; e-mail shaulya{at}post.tau.ac.il; fax
972-3-6409380.
© 2001 American Society of Plant Physiologists
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