Protein Geranylgeranyltransferase I Is Involved in Specific Aspects of Abscisic Acid and Auxin Signaling in Arabidopsis

Abstract

(Q.Z., M.P.R.)Arabidopsis (Arabidopsis thaliana) mutants lacking a functional ERA1 gene, which encodes the b-subunit of protein farnesyltransferase (PFT), exhibit pleiotropic effects that establish roles for protein prenylation in abscisic acid (ABA) signaling and meristem development. Here, we report the effects of T-DNA insertion mutations in the Arabidopsis GGB gene, which encodes the b-subunit of protein geranylgeranyltransferase type I (PGGT I). Stomatal apertures of ggb plants were smaller than those of wild-type plants at all concentrations of ABA tested, suggesting that PGGT I negatively regulates ABA signaling in guard cells. However, germination of ggb seeds in response to ABA was similar to the wild type. Lateral root formation in response to exogenous auxin was increased in ggb seedlings compared to the wild type, but no change in auxin inhibition of primary root growth was observed, suggesting that PGGT I is specifically involved in negative regulation of auxin-induced lateral root initiation. Unlike era1 mutants, ggb mutants exhibited no obvious developmental phenotypes. However, era1 ggb double mutants exhibited more severe developmental phenotypes than era1 mutants and were indistinguishable from plp mutants lacking the shared a-subunit of PFT and PGGT I. Furthermore, overexpression of GGB in transgenic era1 plants partially suppressed the era1 phenotype, suggesting that the relatively weak phenotype of era1 plants is due to partial redundancy between PFT and PGGT I. These results are discussed in the context of Arabidopsis proteins that are putative substrates of PGGT I.Protein prenylation involves the formation of a thioether bond between a farnesyl (C15) or geranylgeranyl (C20) group and a C-terminal Cys residue (Clarke, 1992;Zhang and Casey, 1996). This posttranslational modification promotes protein-membrane and, in many cases, protein-protein interactions. Three protein prenyltransferases are known to catalyze protein prenylation in plants and other eukaryotes: (1) a heterodimeric protein farnesyltransferase (PFT) that consists of a-and b-subunits and transfers a farnesyl group from farnesyl pyrophosphate (FPP) to proteins bearing a C-terminal CaaX motif, where ''C'' is Cys, ''a'' is often an aliphatic amino acid, and ''X'' is generally Met, Ala, Gln, Ser, or Cys; (2) a heterodimeric type I protein geranylgeranyltransferase (PGGT I) that shares a common a-subunit with PFT but possesses a distinct b-subunit and transfers a geranylgeranyl group from geranylgeranyl pyrophosphate (GGPP) to proteins bearing a C-terminal CaaX motif, where ''X'' is Leu; and (3) a heterotrimeric type II protein geranylgeranyltransferase (PGGT II or Rab PGGT) that consists of distinct a-and b-subunits, as well as a third subunit called the Rab escort protein, and transfers a geranylgeranyl group from GGPP to Rab protein substrates (Clarke, 1992;Zhang and Casey, 1996;Crowell, 2000). PFT subunits have been identified and characterized from pea (Pisum sativum; Yang et al., 1993;Qian et al., 1996), tomato (Lycopersicon ...

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

“…The latter exhibits an exaggerated era1-like phenotype, suggesting that PGGT 1 compensates for loss of PFT in era1 mutants. This suggestion was confirmed by suppression of the era1 phenotype in Arabidopsis plants overproducing the PGGT 1 b-subunit (Johnson et al, 2005). Protein isoprenylation is also involved in plant stress responses.…”

“…In addition, two geranylgeranylated proteins, ROP2 and ROP6, have been shown to be involved in negative regulation of ABA signaling (Lemichez et al, 2001;Li et al, 2001;Yang, 2002). Similarly, the existence of geranylgeranylated proteins involved in negative regulation of auxin signaling is inferred from the enhanced response of GERANYLGERANYL TRANSFERASE b (GGB) mutants, which lack the b-subunit of PGGT 1, to auxininduced lateral root initiation (Johnson et al, 2005). Mutants lacking either of two geranylgeranylated G protein g-subunits were subsequently shown to exhibit the same phenotype (Trusov et al, 2007).…”

MUBs, a Family of Ubiquitin-fold Proteins That Are Plasma Membrane-anchored by Prenylation

“…Tissue was chopped in 1 ml/g of extraction buffer (400 mM sucrose, 1 mM Na 4 EDTA, 100 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 2 mM N-ethylmaleimide, and 1ϫ Complete protease inhibitors (Roche Applied Science)) with a razor blade, filtered through Miracloth (EMD Biosciences, San Diego), and then clarified at 500 ϫ g. The supernatants were centrifuged at 100,000 ϫ g for 60 min at 4°C to separate the cytosolic and membrane fractions. The pellets were resuspended in an equal volume of extraction buffer without or with 1% Triton X-114, 1 M NaCl, or 0.1 M sodium carbonate (pH 11), incubated at 0 -4°C for 1 h, and then recentrifuged at 100,000 ϫ g for 1 h. The plp-1, era1-4, and ggb-2 mutants were described previously (26,28,30).…”

Thematic review series: Lipid Posttranslational Modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I

“…Both enzymes have been identified in a number of species, including mammals (17-21), fungi (22-25), plants (26, 27), and protists (28, 29); the genes encoding FTase and GGTase-I have been cloned from several of these species (20,(30)(31)(32)(33)(34)(35). These enzymes have been shown to be essential for the function of these organisms: elimination of these enzymes results in severe defects or lethality in many instances (22,26,(36)(37)(38). …”